U.S. patent application number 10/418251 was filed with the patent office on 2004-04-15 for isolation of a rearranged human immunoglobulin gene from a chimeric mouse and recombinant production of the encoded immunoglobulin.
This patent application is currently assigned to Kirin Beer Kabushiki Kaisha. Invention is credited to Hanaoka, Kazunori, Ishida, Isao, Oshimura, Mitsuo, Tomizuka, Kazuma, Yoshida, Hitoshi.
Application Number | 20040073957 10/418251 |
Document ID | / |
Family ID | 28794711 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040073957 |
Kind Code |
A1 |
Tomizuka, Kazuma ; et
al. |
April 15, 2004 |
Isolation of a rearranged human immunoglobulin gene from a chimeric
mouse and recombinant production of the encoded immunoglobulin
Abstract
The specification relates to a method for producing a chimeric
non-human animal, which comprises preparing a microcell containing
a foreign chromosome(s) or a fragment(s) thereof and transferring
the foreign chromosome(s) or fragment(s) thereof into a pluripotent
cell by fusion with the microcell; a chimeric non-human animal
which can be produced by the above method and its progeny; tissues
and cells derived therefrom; and a method for using the same.
Further, a pluripotent cell containing a foreign chromosome(s) or a
fragment(s) thereof, a method for producing the same, and a method
for using the same are also provided. Moreover, a pluripotent cell
in which at least two endogenous genes are disrupted, and a method
for producing the same by homologous recombination are
provided.
Inventors: |
Tomizuka, Kazuma;
(Yokohama-shi, JP) ; Yoshida, Hitoshi;
(Yokohama-shi, JP) ; Hanaoka, Kazunori;
(Sagamihara-shi, JP) ; Oshimura, Mitsuo;
(Yonago-shi, JP) ; Ishida, Isao; (Yokohama-shi,
JP) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Kirin Beer Kabushiki Kaisha
|
Family ID: |
28794711 |
Appl. No.: |
10/418251 |
Filed: |
November 13, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10418251 |
Nov 13, 2003 |
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09033936 |
Mar 2, 1998 |
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6632976 |
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09033936 |
Mar 2, 1998 |
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PCT/JP96/02427 |
Aug 29, 1996 |
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Current U.S.
Class: |
800/6 ;
800/21 |
Current CPC
Class: |
C07K 16/18 20130101;
C12N 2517/02 20130101; A01K 2217/05 20130101; A01K 2267/01
20130101; A01K 67/0275 20130101; A01K 2227/105 20130101; C07K 16/00
20130101; C12N 2800/30 20130101; A01K 2207/15 20130101; A01K
2217/00 20130101; A01K 67/0276 20130101; A01K 67/0278 20130101;
C07K 2317/21 20130101; A01K 2267/03 20130101; A01K 67/0271
20130101; C12N 15/90 20130101; A01K 2217/075 20130101; C12N 15/8509
20130101 |
Class at
Publication: |
800/006 ;
800/021 |
International
Class: |
A01K 067/027 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 29, 1995 |
JP |
7/24340 |
Feb 15, 1996 |
JP |
8/27940 |
Feb 28, 1997 |
JP |
62309/1997 |
Claims
What is claimed is:
1. A method for producing a chimeric non-human animal, which
comprises preparing a microcell containing a foreign chromosome(s)
or a fragment(s) thereof and transferring the foreign chromosome(s)
or fragment(s) into a pluripotent cell by fusion with the
microcell.
2. A method for producing a pluripotent cell containing a foreign
chromosome(s) or a fragment(s) thereof, which comprises preparing a
microcell containing a foreign chromosome(s) or a fragment(s)
thereof and transferring the foreign chromosome(s) or fragment(s)
thereof into a pluripotent cell by fusion with the microcell.
3. The method of claim 1 or 2, wherein the foreign chromosome(s) or
fragment(s) thereof is(are) larger than 670 kb.
4. The method of claim 3, wherein the foreign chromosome(s) or
fragment(s) thereof is(are) at least 1 Mb in length.
5. The method of claim 1 or 2, wherein the foreign chromosome or
fragment thereof contains a region encoding an antibody.
6. The method of claim 1 or 2, wherein the microcell containing a
foreign chromosome(s) or a fragment(s) thereof is induced from a
hybrid cell prepared by the fusion of a cell from which the foreign
chromosome(s) or fragment(s) thereof is(are) derived, with a cell
having a high ability to form a microcell.
7. The method of claim 6, wherein the microcell containing a
foreign chromosome(s) or a fragment(s) thereof is induced from a
cell prepared by a further fusion of the microcell induced from
said hybrid cell with a cell having a high ability to form a
microcell.
8. The method of claim 6, wherein the cell from which the foreign
chromosome(s) or fragment(s) thereof is(are) derived is a human
normal diploid cell.
9. The method of any one of claims 6-8, wherein the cell having a
high ability to form a microcell is a mouse A9 cell.
10. The method of claim 1 or 2, wherein the pluripotent cell is one
selected from embryonal carcinoma cells, embryonic stem cells,
embryonic germ cells and mutants thereof.
11. The method of claim 1 or 2, wherein the foreign chromosome or
fragment thereof contains a gene of interest and the pluripotent
cell has a disrupted gene identical with or homologous to said gene
of interest on the foreign chromosome or fragment thereof.
12. The method of claim 11, wherein the foreign chromosome or
fragment thereof contains at least two genes of interest and the
pluripotent cell has disrupted genes identical with or homologous
to said genes of interest on the foreign chromosome or fragment
thereof.
13. The method of claim 11, wherein one or both alleles of a gene
identical with or homologous to the gene of interest on the foreign
chromosome or fragment thereof are disrupted in the pluripotent
cell.
14. The method of claim 11, wherein the gene of interest is an
antibody gene.
15. The method of claim 14, wherein the antibody gene is one or
more sets of antibody heavy-chain and light-chain genes.
16. The method of claim 1, wherein the foreign chromosome or
fragment thereof contains a gene of interest and said foreign
chromosome or fragment thereof is transferred into a pluripotent
cell having a disrupted gene identical with or homologous to said
gene of interest and then, a chimera is produced from the
pluripotent cell by using an embryo of a non-human animal in a
strain deficient in an endogenous gene identical with or homologous
to said gene of interest.
17. The method of claim 16, wherein the non-human animal in a
strain deficient in an endogenous gene identical with or homologous
to the gene of interest is produced by homologous recombination in
gene targeting.
18. The method of claim 1, wherein the chimeric non-human animal
retains the foreign chromosome(s) or fragment(s) thereof, expresses
the gene(s) on the foreign chromosome(s) or fragment(s) thereof,
and can transmit the foreign chromosome(s) or fragment(s) thereof
to its progeny.
19. The method of claim 1, wherein the chimeric non-human animal is
a mammal.
20. The method of claim 19, wherein the mammal is a mouse.
21. A pluripotent cell containing a foreign chromosome(s) or a
fragment(s) thereof.
22. The cell of claim 21, wherein the foreign chromosome(s) or
fragment(s) thereof is(are) larger than 670 kb.
23. The cell of claim 21, wherein the foreign chromosome or
fragment thereof contains a gene of interest and the pluripotent
cell has a disrupted endogenous gene identical with or homologous
to said gene of interest on the foreign chromosome or a fragment
thereof.
24. The cell of claim 23, wherein the foreign chromosome or
fragment thereof contains at least two genes of interest and the
pluripotent cell has disrupted endogenous genes identical with or
homologous to said genes of interest on the foreign chromosome or a
fragment thereof.
25. The cell of claim 23, wherein one or both alleles of an
endogenous gene identical with or homologous to the gene of
interest are disrupted in the pluripotent cell.
26. The cell of claim 21, wherein the foreign chromosome or
fragment thereof contains an antibody gene.
27. The cell of claim 26, wherein the antibody gene is one or more
sets of antibody heavy-chain and light-chain genes.
28. The cell of claim 21, wherein the pluripotent cell is one
selected from embryonal carcinoma cells, embryonic stem cells,
embryonic germ cells and mutants thereof.
29. A chimeric non-human animal retaining a foreign chromosome(s)
or a fragment(s) thereof and expressing a gene(s) on the foreign
chromosome(s) or fragment(s) thereof, or its progeny retaining the
foreign chromosome(s) or fragment(s) thereof and expressing the
gene(s) on the foreign chromosome(s) or fragment(s) thereof.
30. The chimeric non-human animal or its progeny of claim 29,
wherein the foreign chromosome(s) or fragment(s) thereof is(are)
larger than 670 kb.
31. The chimeric non-human animal or its progeny of claim 29,
wherein the foreign chromosome or fragment thereof contains a gene
of interest and the animal has a disrupted endogenous gene
identical with or homologous to said gene of interest.
32. The chimeric non-human animal or its progeny of claim 31,
wherein the foreign chromosome or fragment thereof contains at
least two genes of interest and said animal has disrupted
endogenous genes identical with or homologous to said genes of
interest.
33. The chimeric non-human animal or its progeny of claim 31,
wherein one or both alleles of an endogenous gene identical with or
homologous to said gene of interest are disrupted.
34. The chimeric non-human animal or its progeny of claim 31,
wherein the gene of interest is an antibody gene.
35. The chimeric non-human animal or its progeny of claim 34,
wherein the antibody gene is one or more sets of antibody
heavy-chain and light-chain genes.
36. A non-human animal which can be produced by mating the chimeric
non-human animals or their progenies of claim 29, said non-human
animal retaining the foreign chromosome(s) or fragment(s) thereof
and expressing the gene(s) on the foreign chromosome(s) or
fragment(s) thereof, or its progeny retaining the foreign
chromosome(s) or fragment(s) thereof and expressing the gene(s) on
the foreign chromosome(s) or fragment(s) thereof.
37. A non-human animal retaining the foreign chromosome(s) or
fragment(s) thereof and expressing a gene(s) on the foreign
chromosome(s) or fragment(s) thereof, which can be produced by
mating the chimeric non-human animal or its progeny of claim 29, or
the non-human animal or its progeny of claim 36, with a non-human
animal in a strain deficient in said gene(s) or a gene homologous
thereto, or its progeny retaining the foreign chromosome(s) or
fragment(s) thereof and expressing the gene(s) on the foreign
chromosome(s) or fragment(s) thereof.
38. A tissue from the chimeric non-human animal or its progeny of
claim 29 or from the non-human animal or its progeny of claim 36 or
from the non-human animal or its progeny of claim 37.
39. A cell from the chimeric non-human animal or its progeny of
claim 29 or from the non-human animal or its progeny of claim 36 or
from the non-human animal or its progeny of claim 37.
40. The cell of claim 39, which is a B cell.
41. A hybridoma prepared by the fusion of the cell of claim 40 with
a myeloma cell.
42. A method for producing a biologically active substance, which
comprises expressing the gene(s) on the foreign chromosome(s) or
fragment(s) thereof in the chimeric non-human animal or its progeny
of claim 29, the non-human animal or its progeny of claim 36 or the
non-human animal or its progeny of claim 37, or a tissue or a cell
thereof, and recovering the biologically active substance as an
expression product.
43. The method of claim 42, wherein the cell of the chimeric
non-human animal is a B cell.
44. The method of claim 43, wherein the B cell is immortalized by
fusion with a myeloma cell.
45. The method of claim 42, wherein the biologically active
substance is an antibody.
46. The method of claim 45, wherein the antibody is an antibody of
a mammal.
47. The method of claim 46, wherein the antibody of a mammal is a
human antibody.
48. A biologically active substance which can be produced by the
method of claim 42.
49. A non-human animal retaining at least one human antibody gene
larger than 670 kb and expressing the gene.
50. The non-human animal of claim 49, which retains at least one
human antibody gene of at least 1 Mb and expresses the gene.
51. The non-human animal of claim 49, wherein the human antibody
gene is selected from the group consisting of human heavy-chain
gene, human light-chain .kappa. gene, human light-chain .lambda.
gene, and combinations thereof.
52. The non-human animal of claim 49, which is deficient in a
non-human animal antibody gene identical with or homologous to the
human antibody gene.
53. The non-human animal of claim 52, wherein the deficiency of
said non-human animal antibody gene is caused by disrupting the
non-human animal antibody gene by homologous recombination.
54. A hybridoma prepared by the fusion of a spleen cell of the
non-human animal of claim 49 with a myeloma cell.
55. An antibody produced by the hybridoma of claim 54.
56. A non-human animal expressing at least one class or subclass of
human antibody.
57. The non-human animal of claim 56, which is deficient in an
endogenous antibody gene identical with or homologous to the
expressed class or subclass of human antibody gene.
58. The non-human animal of claim 56, wherein the class or subclass
of human antibody is selected from IgM, IgG, IgE, IgA, IgD and
their subclasses, and combinations thereof.
59. A non-human animal retaining a foreign DNA(s) larger than 670
kb and expressing a gene(s) on the foreign DNA(s).
60. The non-human animal of claim 59, which is deficient in an
endogenous gene identical with or homologous to the expressed gene
on the foreign DNA.
61. The non-human animal of claim 59 which retains a foreign DNA(s)
of at least 1 Mb and expresses the gene(s) on the foreign
DNA(s).
62. The non-human animal of claim 61, which is deficient in an
endogenous gene identical with or homologous to the expressed gene
on the foreign DNA.
63. A method for producing a transgenic non-human animal, which
comprises preparing a microcell containing a foreign chromosome(s)
or a fragment(s) thereof, transferring the foreign chromosome(s) or
fragment(s) into a cultured cell derived from a blastcyst by fusion
with the microcell and transplanting the nucleus of the cultured
cell into an enucleated unfertilized egg.
64. A pluripotent cell in which at least two endogenous genes are
disrupted.
65. The cell of claim 64, in which each of the endogenous genes is
disrupted in one or both alleles.
66. The cell of claim 64, wherein the disrupted endogenous genes
are antibody genes.
67. The cell of claim 66, wherein the antibody genes are antibody
heavy-chain and light-chain genes.
68. The cell of claim 64, wherein the pluripotent cell is one
selected from embryonal carcinoma cells, embryonic stem cells,
embryonic germ cells and mutants thereof.
69. A method of producing the cell of claim 64 by at least two
homologous recombinations.
70. The method of claim 69, which comprises the steps of:
disrupting one allele of the endogenous gene in the pluripotent
cell by homologous recombination using a drug-resistant marker
gene; culturing the pluripotent cell in the presence of the drug to
select drug-resistant cells; and screening the selected
drug-resistant cells to yield a cell in which both alleles of the
endogenous gene have been disrupted.
71. The method of claim 69, in which one allele of the endogenous
gene in the pluripotent cell is disrupted by homologous
recombination using a drug-resistant marker gene and the other
allele of the endogenous gene is disrupted by another homologous
recombination using a drug-resistant marker gene.
72. The method of claim 71, wherein the same drug-resistant marker
gene is used in the two homologous recombinations.
73. The method of claim 71, wherein different drug-resistant marker
genes are used in the two homologous recombinations.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation-in-part application of International
Application No. PCT/JP96/02427 with an international filing date of
Aug. 29, 1996.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to chimeric non-human animals,
a method for producing the same and a method for using the same.
The present invention allows chimeric non-human animals to retain a
foreign giant DNA fragment(s) of at least 1 Mb and to express the
gene(s) on such a fragment(s), which was impossible heretofore.
Hence, the following becomes possible by using the method.
[0003] Production of animals which retain and express a full length
of a gene encoding a biologically active substance, for example, a
full length of human antibody gene. The biologically active
substance, for example, a human-type antibody is useful as a
pharmaceutical product.
[0004] Analysis of functions of human giant genes (e.g.,
histocompatibility antigen, dystrophin, etc.) in animals.
[0005] Production of model animals with human dominant hereditary
disease and a disease due to chromosomal aberration.
[0006] The present invention relates to pluripotent cells in which
endogenous genes are disrupted, use of the same, and a method for
producing chimeric non-human animals and use of the animals. If a
foreign chromosome or a fragment thereof containing a gene encoding
a gene product identical with or homologous to the gene product
encoded by the disrupted endogenous gene is transferred into the
pluripotent cell of the present invention as a recipient cell so
that a desired functional cell or a desired chimeric non-human
animal is produced from the cell, the transferred gene can be
expressed efficiently without differentiation of the pluripotent
cell into a germ cell. Even if a germ cell of the non-human animal
is affected or the pluripotent cell cannot be differentiated into a
germ cell by the disruption of the endogenous gene or the
introduction of a foreign gene, a functional cell, or a chimeric
non-human animal, a tissue or a cell of the animal can retain and
express a foreign giant DNA fragment in excess of the heretofore
unattainable 1 Mb (a million bases) in conditions of a deficiency
in the endogenous gene and a decrease in the production of an
endogenous gene product by producing the desired functional cell or
non-human animal from the pluripotent cell.
[0007] Techniques of expressing foreign genes in animals, that is,
techniques of producing transgenic animals are used not only for
obtaining information on the gene's functions in living bodies but
also for identifying DNA sequences that regulate the expression of
the genes (e.g., Magram et al., Nature, 315:338, 1985), for
developing model animals with human diseases (Yamamura et al.,
"Manual of model mice with diseases" published by Nakayama Shoten,
1994), for breeding farm animals (e.g., Muller et al., Experientia,
47:923, 1991) and for producing useful substances with these
animals (e.g., Velander et al., P.N.A.S., 89:12003, 1992). Mice
have been used the most frequently as hosts for gene transfer.
Since mice have been studied in detail as experimental animals and
the embryo manipulating techniques for mice have been established,
they are the most appropriate kind of mammals for gene
transfer.
[0008] Two methods are known for transferring foreign genes into
mice. One is by injecting DNA into a pronucleus of a fertilized egg
(Gordon et al., P.N.A.S., 77:7380, 1980). The other is by
transferring DNA into a pluripotent embryonic stem cell
(hereinafter referred to as "ES cell") to produce a chimeric mouse
(Takahashi et al., Development, 102:259, 1988). In the latter
method, the transferred gene is retained only in ES
cell-contributing cells and tissues of chimeric mice whereas it is
retained in all cells and tissues of progenies obtained via ES
cell-derived germ cells. These techniques have been used to produce
a large number of transgenic mice up to now.
[0009] However, there had been a limit of the size of DNA capable
of being transferred and this restricts the application range of
these techniques. The limit depends on the size of DNA which can be
cloned. One of the largest DNA fragments which have ever been
transferred is a DNA fragment of about 670 kb cloned into a yeast
artificial chromosome (YAC) (Jakobovits et al., Nature, 362:255,
1993). Recently, introduction of YAC containing an about 1 Mb DNA
fragment containing about 80 percent of variable regions and
portions of constant regions (C.mu., C.delta. and C.gamma.) of a
human antibody heavy-chain was reported (Mendes et al., Nature
Genetics, 15:146, 1997). These experiments were carried out by
fusing a YAC-retaining yeast cell with a mouse ES cell. Although it
is believed that foreign DNA of up to about 2 Mb can be cloned on
YAC (Den Dunnen et al., Hum. Mol. Genet., 1:19, 1992), the
recombination between homologous DNA sequences occurs frequently in
budding yeast cells and therefore, in some cases, a human DNA
fragment containing a large number of repeated sequences is
difficult to retain in a complete form. In fact, certain
recombinations occur in 20-40% of the clones of YAC libraries
containing human genomic DNA (Green et al., Genomics, 11:584,
1991).
[0010] In another method that was attempted, a metaphase chromosome
from a cultured human cell was dissected under observation with a
microscope and the fragment (presumably having a length of at least
10 Mb) was injected into a mouse fertilized egg (Richa et al.,
Science, 245:175, 1989). In the resulting mice, a human specific
DNA sequence (Alu sequence) was detected but the expression of
human gene was not confirmed. In addition, the procedure used in
this method to prepare chromosomes causes unavoidable fragmentation
of DNA into small fragments due to the use of acetic acid and
methanol in fixing the chromosome on slide glass and the
possibility that the injected DNA exists as an intact sequence is
small.
[0011] In any event, no case has been reported to date that
demonstrates successful transfer and expression in mice of
uninterrupted foreign DNA fragments having a length of at least 1
Mb.
[0012] Useful and interesting human genes which are desirably
transferred into mice, such as genes for antibody (Cook et al.,
Nature Genetics, 7: 162, 1994), for T cell receptor (Hood et al.,
Cold Spring Harbor Symposia on Quantitative Biology, Vol. LVIII,
339, 1993), for histocompatibility antigen (Carrol et al., P.N.A.S,
84:8535, 1987), for dystrophin (Den Dunnen et al., supra) are known
to be such that their coding regions have sizes of at least 1 Mb.
Since human-type antibodies are important as pharmaceutical
products, the production of mice which retain and express full
lengths of genes for human immunoglobulin heavy chains (.about.1.5
Mb, Cook et al., supra), and light chain .kappa. (.about.3 Mb,
Zachau, Gene, 135:167, 1993), and light chain .lambda. (.about.1.5
Mb, Frippiat et al., Hum. Mol. Genet., 4:983, 1995) is desired but
this is impossible to achieve by the state-of-the-art technology
(Nikkei Biotec, Jul. 5, 1993).
[0013] Many of the causative genes for human dominant hereditary
disease and chromosomal aberration which causes congenital
deformity (Down's syndrome, etc.) have not been cloned and only the
information on the approximate location of the genes on chromosome
is available. For example, when a gene of interest is found to be
located on a specific G band, which is made visible by subjecting a
metaphase chromosome to Giemsa staining, the G band has usually a
size of at least several Mb to 10 Mb. In order to transfer these
abnormal phenotypes into mice, it is necessary to transfer
chromosomal fragments of at least several Mb that surround the
causative genes, but this is also impossible with the presently
available techniques.
[0014] Hence, it is desired to develop a technique by which a
foreign DNA longer than the heretofore critical 1 Mb can be
transferred into a mouse and expressed in it.
[0015] DNA longer than 1 Mb can be transferred into cultured animal
cells by the techniques available today. Such transfer is carried
out predominantly by using a chromosome as a mediator. In the case
of human, chromosomes have sizes of about 50-300 Mb. Some methods
for chromosome transfer into cells have been reported (e.g.,
McBride et al., P.N.A.S., 70:1258, 1973). Among them, microcell
fusion (Koi et al., Jpn. J. Cancer Res., 80:413, 1989) is the best
method for selective transfer of a desired chromosome. The
microcell is a structural body in which one to several chromosomes
are encapsulated with a nuclear membrane and a plasma membrane. A
few chromosomes (in many cases, one chromosome) can be transferred
by inducing a microcell with an agent that inhibits the formation
of spindle in a specific kind of cell, separating the microcell and
fusing it with a recipient cell. The resulting libraries of
monochromosomal hybrid cells containing only one human chromosome
have been used for mapping known genes and specifying the
chromosomes on which unknown tumor-suppressor genes and cellular
senescence genes exist (e.g., Saxon et al., EMBO J., 5:3461, 1986).
In addition, it is possible to fragment a chromosome by irradiating
a microcell with .gamma.-rays and to transfer part of the fragments
(Koi et al., Science, 260:361, 1993). As described above, microcell
fusion is considered to be an appropriate method for transferring
DNA larger than 1 Mb into a cultured animal cell.
[0016] The expectation that a mouse could be generated from a
cultured cell turned to a real fact when the ES cell which has
stable pluripotency was discovered (Evans et al., Nature, 292:154,
1981). Foreign genes, various mutations and mutations by targeted
gene recombination could be introduced into the ES cell, making it
possible to perform a wide variety of genetic modifications in mice
(e.g., Mansour et al., Nature, 336:348, 1988). The ES cell can be
used to produce a mouse having a disrupted target gene by gene
targeting techniques. The mouse is mated with a transgenic mouse
having a gene of interest to produce a mouse that expresses the
gene of interest efficiently. For example, a mouse having a
disrupted endogenous antibody gene can be mated with a mouse having
a human antibody gene transferred to produce a mouse that expresses
the human antibody efficiently. A normal diploid cell has alleles.
A transgenic mouse having one allele of an mouse antibody
heavy-chain gene disrupted expresses an increased level of human
antibody in its serum. A mouse having both alleles of mouse
antibody heavy-chain gene disrupted expresses a further remarkably
increased level of human antibody (S. D. Wagner et al., Genomics,
35:405-414, 1996).
[0017] Some researchers have developed a technique in which one
allele of a target gene is disrupted, and then the concentration of
a selective drug is increased, thereby deleting both alleles of the
target gene (double knock-out). However, this technique holds the
possibility of a decrease in the ability of the target
gene-deficient cell to differentiate into a germ cell because the
target gene-deficient cell obtained by the
high-concentration-selective-culture method is cultured in vivo for
a long period and because the drug-selection pressure is severe
(Takatsu.cndot.Taki, Experimental Medicine, supplement, Biomanual
UP Series Basic Techniques for Immunological Study, Yodo-sha,
1995). In another case, if two kinds of selective drugs are used
for double knocking-out, for example, if a neomycin-resistant cell
is subjected to a double knock-out treatment with hygromycin, the
double drug-resistant ES cell is rarely differentiated to produce a
mutant mouse (Watanabe et al., Tissue Culture 21, 42-45, 1995). ES
cells may lose their differentiation and growth capabilities under
certain culture conditions. When a gene targeting procedure is
performed twice, ES cells do not lose the ability to differentiate
into germ cells of a chimeric mouse but the second homologous
recombination frequency is extremely low (Katsuki et al.,
Experimental Medicine, Vol. 11, No. 20, special number, 1993).
Hence, when a target gene-deficient homozygote is produced,
particularly when at least two target genes are targeted, a mouse
deficient in each target gene is produced and then the produced
mice are mated with each other to produce a homozygote mouse
deficient in at least two genes (N. Longberg et al., Nature,
368:856-859, 1994). If genes to be disrupted exist close to each
other and if a mouse deficient in at least two genes cannot be
obtained by mating, heterozygote mice deficient in the two target
genes are produced from ES cells and they are mated to produce
homodeficient mice (J. H. van Ree et al., Hum Mol Genet
4:1403-1409, 1995).
[0018] An attempt to differentiate a pluripotent ES cell into a
functional cell in vitro has been made (T. Nakano et al., Science,
265:1098-1101, 1994, A. J. Potocnik et al., The EMBO Journal,
13:5274-5283, 1994). The cultivation system used in this attempt,
for example, a system in which the differentiation into a mature B
cell can be induced is expected to be used in the identification of
unknown growth and differentiation factors which will work in
development and differentiation processes of B cells.
[0019] As long as the transfer of giant DNA is concerned, it has
been believed that the size of the aforementioned foreign DNA
fragment which can be cloned into a YAC vector is the upper limit.
The prior art technology of chromosome transfer for introducing a
longer DNA into cultured cells has never been applied to gene
transfer into mice and this has been believed to be difficult to
accomplish (Muramatsu et al., "Transgenic Biology", published by
Kodansha Scientific, p.143-, 1989).
[0020] The reasons are as follows.
[0021] The transfer of a human chromosome into a mouse ES cell of a
normal karyotype as a recipient cell would be a kind of transfer of
chromosomal aberration. Up to now, it has been believed that
genetic aberration at chromosomal levels which is large enough to
be recognizable with microscopes is generally fatal to the
embryogeny in mice (Gropp et al., J. Exp. Zool., 228:253, 1983 and
Shinichi Aizawa, "Biotechnology Manual Series 8, Gene Targeting",
published by Yodosha, 1995).
[0022] Available human chromosomes are usually derived from
finitely proliferative normal fibroblasts or differentiated somatic
cells such as cancer cells and the like. It was believed that if a
chromosome derived from such a somatic cell was transferred into an
undifferentiated ES cell, the transferred chromosome might cause
differentiation of the ES cell or its senescence (Muller et al.,
Nature, 311:438, 1984; Sugawara, Science, 247:707, 1990).
[0023] Only few studies have been reported as to whether a somatic
cell-derived chromosome introduced into an early embryo can
function in the process of embryonic development as normally as a
germ cell-derived chromosome to ensure the expression of a specific
gene in various kinds of tissues and cells. One of the big
differences between the two chromosomes is assumed to concern
methylation of the chromosomal DNA. The methylation is changed
according to differentiation of cells and its important role in the
expression of tissue-specific genes has been suggested (Ceder,
Cell, 53:3, 1988). For example, it has been reported that if a
methylated DNA substrate is introduced into a B cell, the
methylated DNA is maintained after replication and suppresses a
site-directed recombination reaction which is essential to the
activation of an antibody gene (Hsieh et al., EMBO J., 11:315,
1992). In addition, it was reported that higher levels of de novo
methylation occurred in established cell lines than in vivo
(Antequera et al., Cell, 62:503, 1990). On the basis of the studies
reported, it could not be easily expected that an antibody gene in
a human fibroblast or a human-mouse hybrid cell which was likely to
be methylated at a high level would be normally expressed in a
mouse B cell.
[0024] It should be noted that there are two related reports of
Illmensee et al. (P.N.A.S., 75:1914, 1978; P.N.A.S., 76:879, 1979).
One report is about the production of chimeric mice from fused
cells obtained by fusing a human sarcoma cell with a mouse EC cell
and the other is about the production of chimeric mice from fused
cells obtained by fusing a rat liver cancer cell with a mouse EC
cell. Many questions about the results of the experiments in these
two reports were pointed out and thus these reports are considered
unreliable (Noguchi et al., "Mouse Teratoma", published by
Rikogakusha, Section 5, 1987). Although it has been desired to
perform a follow-up as early as possible, as of today when 17 years
have passed since the publication of these reports, successful
reproduction of these experiments has not been reported. Hence, it
is believed that foreign chromosomes cannot be retained and the
genes on the chromosomes cannot be expressed in mice by the method
described in these reports.
[0025] Under these circumstances, it has been believed to be
difficult to transfer a giant DNA such as a chromosomal fragment
and express it in an animal such as mouse. Actually, no study has
been made about this problem since the Illmensee's reports.
[0026] Therefore, an object of the present invention is to provide
chimeric non-human animals which retain foreign chromosomes or
fragments thereof and express genes on the chromosomes or
fragments, and their progenies, and a method for producing the
same.
[0027] It is also an object of the present invention to provide
pluripotent cells containing foreign chromosomes or fragments
thereof and a method for producing the pluripotent cells.
[0028] Another object of the present invention is to provide
tissues and cells derived from the chimeric non-human animals and
their progenies.
[0029] A further object of the present invention is to provide
hybridomas prepared by fusing the cells derived from the chimeric
non-human animals and their progenies with myeloma cells.
[0030] A still further object of the present invention is to
provide a method for producing a biologically active substance that
is an expression product of the gene on a foreign chromosome or a
fragment thereof by using the chimeric non-human animals or their
progenies, or their tissues or cells.
[0031] It is also an object of the present invention to provide
pluripotent cells which can be used as recipient cells into which a
foreign chromosome(s) or a fragment(s) thereof is transfered in the
production of chimeric non-human animals retaining the foreign
chromosome(s) or fragment(s) thereof and expressing a gene(s) on
the foreign chromosome(s) or fragment(s) thereof.
[0032] A further object of the present invention is to provide a
method for using the pluripotent cells.
SUMMARY OF THE INVENTION
[0033] As a result of the various studies conducted to achieve the
above objects, the inventors succeeded in transferring chromosomes
or fragments thereof derived from human normal fibroblast cells
into mouse ES cells and obtaining clones which were capable of
stable retention of the chromosomes or fragments. Moreover, they
produced from these ES clones those chimeric mice which retained
human chromosomes in normal tissues and which expressed several
human genes including human antibody heavy-chain genes. It has
become possible to make that animals retain and express giant DNA
fragments by the series of these techniques, although this has been
impossible by conventional techniques. Moreover, the inventors
succeeded in obtaining embryonic stem cells having both of antibody
heavy-chain and light-chain genes knocked out.
[0034] The subject matter of the present invention is as
follows:
[0035] 1. A method for producing a chimeric non-human animal, which
comprises preparing a microcell containing a foreign chromosome(s)
or a fragment(s) thereof and transferring the foreign chromosome(s)
or fragment(s) into a pluripotent cell by fusion with the
microcell.
[0036] 2. A method for producing a pluripotent cell containing a
foreign chromosome(s) or a fragment(s) thereof, which comprises
preparing a microcell containing a foreign chromosome(s) or a
fragment(s) thereof and transferring the foreign chromosome(s) or
fragment(s) thereof into a pluripotent cell by fusion with the
microcell.
[0037] In the method of item 1 or 2, the foreign chromosome(s) or
fragment(s) thereof may be larger than 670 kb, further, at least 1
Mb (one million base pairs). The foreign chromosome or fragment
thereof may contain a region encoding an antibody. The microcell
containing a foreign chromosome(s) or a fragment(s) thereof may be
induced from a hybrid cell prepared by the fusion of a cell from
which the foreign chromosome(s) or fragment(s) thereof is(are)
derived, with a cell having a high ability to form a microcell. The
microcell containing a foreign chromosome(s) or a fragment(s)
thereof may be induced from a cell prepared by a further fusion of
the microcell induced from the hybrid cell with a cell having a
high ability to form a microcell. The cell from which the foreign
chromosome(s) or fragment(s) thereof is(are) derived may be a human
normal diploid cell. The cell having a high ability to form a
microcell may be a mouse A9 cell. The pluripotent cell can be
selected from embryonal carcinoma cells, embryonic stem cells,
embryonic germ cells and mutants thereof. It is preferred that the
foreign chromosome or fragment thereof contains a gene of interest
and that the pluripotent cell has a disrupted gene identical with
or homologous to said gene of interest on the foreign chromosome or
fragment thereof. It is also preferred that the foreign chromosome
or fragment thereof contains at least two genes of interest and
that the pluripotent cell has disrupted genes identical with or
homologous to said genes of interest on the foreign chromosome or
fragment thereof. In the pluripotent cell, one or both alleles of a
gene identical with or homologous to the gene of interest on the
foreign chromosome or fragment thereof may be disrupted. The gene
of interest may be an antibody gene. The antibody gene may be one
or more sets of antibody heavy-chain and light-chain genes. In the
method of item 1 or 2, it is preferred that the foreign chromosome
or fragment thereof contains a gene of interest and that the
foreign chromosome or fragment thereof is transferred into a
pluripotent cell having a disrupted gene identical with or
homologous to the gene of interest and then, a chimera is produced
from the pluripotent cell by using an embryo of a non-human animal
in a strain deficient in an endogenous gene identical with or
homologous to the gene of interest. The non-human animal in a
strain deficient in an endogenous gene identical with or homologous
to the gene of interest can be produced by homologous recombination
in gene targeting. Preferably, the chimeric non-human animal
retains the foreign chromosome(s) or fragment(s) thereof, expresses
the gene(s) on the foreign chromosome(s) or fragment(s) thereof,
and can transmit the foreign chromosome(s) or fragment(s) thereof
to its progeny. The chimeric non-human animal is preferably a
mammal, more preferably a mouse.
[0038] 3. A pluripotent cell containing a foreign chromosome(s) or
a fragment(s) thereof.
[0039] In the pluripotent cell, the foreign chromosome(s) or
fragment(s) thereof may be larger than 670 kb. In the cell of item
3, the foreign chromosome or fragment thereof may contain a gene of
interest and the pluripotent cell has a disrupted gene identical
with or homologous to the gene of interest on the foreign
chromosome or a fragment thereof. The foreign chromosome or
fragment thereof may contain at least two genes of interest and the
pluripotent cell has disrupted genes identical with or homologous
to the genes of interest on the foreign chromosome or a fragment
thereof. In the pluripotent cell, one or both alleles of a gene
identical with or homologous to the gene of interest may be
disrupted. The foreign chromosome or fragment thereof may contain
an antibody gene. The antibody gene may be one or more sets of
antibody heavy-chain and light-chain genes. The pluripotent cell
can be selected from embryonal carcinoma cells, embryonic stem
cells, embryonic germ cells and mutants thereof.
[0040] 4. A chimeric non-human animal retaining a foreign
chromosome(s) or a fragment(s) thereof and expressing a gene(s) on
the foreign chromosome(s) or fragment(s) thereof, or its progeny
retaining the foreign chromosome(s) or fragment(s) thereof and
expressing the gene(s) on the foreign chromosome(s) or fragment(s)
thereof.
[0041] In the chimeric non-human animal or its progeny, the foreign
chromosome(s) or fragment(s) thereof may be larger than 670 kb. The
foreign chromosome or fragment thereof may contain a gene of
interest and the animal may have a disrupted gene identical with or
homologous to the gene of interest. The foreign chromosome or
fragment thereof may contain at least two genes of interest and the
animal may have disrupted genes identical with or homologous to
said genes of interest. In the chimeric non-human animal or its
progeny, one or both alleles of a gene identical with or homologous
to the gene of interest may be disrupted. The gene of interest may
be an antibody gene. The antibody gene may be one or more sets of
antibody heavy-chain and light-chain genes.
[0042] 5. A non-human animal which can be produced by mating the
chimeric non-human animals or their progenies of item 4, said
non-human animal retaining the foreign chromosome(s) or fragment(s)
thereof and expressing the gene(s) on the foreign chromosome(s) or
fragment(s) thereof, or its progeny retaining the foreign
chromosome(s) or fragment(s) thereof and expressing the gene(s) on
the foreign chromosome(s) or fragment(s) thereof.
[0043] 6. A non-human animal retaining the foreign chromosome(s) or
fragment(s) thereof and expressing a gene(s) on the foreign
chromosome(s) or fragment(s) thereof, which can be produced by
mating the chimeric non-human animal or its progeny of item 4, or
the non-human animal or its progeny of item 5, with a non-human
animal in a strain deficient in said gene(s) or a gene homologous
thereto, or its progeny retaining the foreign chromosome(s) or
fragment(s) thereof and expressing the gene(s) on the foreign
chromosome(s) or fragment(s) thereof.
[0044] 7. A tissue from the chimeric non-human animal or its
progeny of item 4 or from the non-human animal or its progeny of
item 5 or from the non-human animal or its progeny of item 6.
[0045] 8. A cell from the chimeric non-human animal or its progeny
of item 4 or from the non-human animal or its progeny of item 5 or
from the non-human animal or its progeny of item 6.
[0046] The cell may be a B cell, a primary culture cell derived
from an animal tissue or a cell fused with an established cell.
[0047] 9. A hybridoma prepared by the fusion of the B cell with a
myeloma cell.
[0048] 10. A method for producing a biologically active substance,
which comprises expressing the gene(s) on the foreign chromosome(s)
or fragment(s) thereof in the chimeric non-human animal or its
progeny of item 4, the non-human animal or its progeny of item 5 or
the non-human animal or its progeny of item 6, or a tissue or a
cell thereof, and recovering the biologically active substance as
an expression product.
[0049] In the method, the cell of the chimeric non-human animal may
be a B cell. The B cell may be immortalized by fusion with a
myeloma cell. The chimeric non-human animal cell may be fused with
a primary culture cell derived from an animal tissue or fused with
an established cell line. The biologically active substance may be
an antibody. The antibody is preferably an antibody of a mammal,
more preferably a human antibody.
[0050] 11. A biologically active substance which can be produced by
the method of item 10.
[0051] 12. A non-human animal retaining at least one human antibody
gene larger than 670 kb and expressing the gene.
[0052] The non-human animal of item 12 preferably retains at least
one human antibody gene of at least 1 Mb and expresses the gene.
The human antibody gene may be a human heavy-chain gene, a human
light-chain .kappa. gene, a human light-chain A gene, or a
combination thereof. The non-human animal of item 12 may be
deficient in a non-human animal antibody gene identical with or
homologous to the human antibody gene. The deficiency of non-human
animal antibody gene may be caused by disrupting the non-human
animal antibody gene by homologous recombination.
[0053] 13. A hybridoma prepared by the fusion of a spleen cell of
the non-human animal of item 12 with a myeloma cell.
[0054] 14. An antibody produced by the hybridoma of item 13.
[0055] 15. A non-human animal expressing at least one class or
subclass of human antibody.
[0056] The non-human animal of item 15 may be deficient in an
endogenous antibody gene identical with or homologous to the
expressed human antibody gene. The class or subclass of human
antibody may be IgM, IgG, IgE, IgA, IgD or a subclass, or a
combination thereof.
[0057] 16. A non-human animal retaining a foreign DNA(s) larger
than 670 kb and expressing a gene(s) on the foreign DNA(s).
[0058] The non-human animal of item 16 may be deficient in an
endogenous gene identical with or homologous to the expressed gene
on the foreign DNA. The non-human animal of item 16 may retain a
foreign DNA(s) of at least 1 Mb and express the gene(s) on the
foreign DNA(s). The non-human animal may be deficient in an
endogenous gene identical with or homologous to the expressed gene
on the foreign DNA.
[0059] 17. A method for producing a transgenic non-human animal,
which comprises preparing a microcell containing a foreign
chromosome(s) or a fragment(s) thereof, transferring the foreign
chromosome(s) or fragment(s) into a cultured cell derived from a
blastcyst by fusion with the microcell and transplanting the
nucleus of the cultured cell into an enucleated unfertilized
egg.
[0060] 18. A pluripotent cell in which at least two endogenous
genes are disrupted.
[0061] In the cell of item 18, each of the endogenous genes may be
disrupted in one or both alleles. The disrupted endogenous genes
may be antibody genes. The disrupted antibody genes may be antibody
heavy-chain and light-chain genes. The pluripotent cell can be
selected from embryonal carcinoma cells, embryonic stem cells,
embryonic germ cells and mutants thereof.
[0062] 19. A method of producing the cell of item 18 by at least
two homologous recombinations.
[0063] The method of item 19 may comprise the steps of:
[0064] disrupting one allele of the endogenous gene in the
pluripotent cell by homologous recombination using a drug-resistant
marker gene;
[0065] culturing the pluripotent cell in the presence of the drug
to select drug-resistant cells; and
[0066] screening the selected drug-resistant cells to yield a cell
in which both alleles of the endogenous gene have been
disrupted.
[0067] In the method of item 19, one allele of the endogenous gene
in the pluripotent cell may be disrupted by homologous
recombination using a drug-resistant marker gene and the other
allele of the endogenous gene may be disrupted by another
homologous recombination using a drug-resistant marker gene. The
same drug-resistant marker gene may be used in the two homologous
recombinations. Alternatively, different drug-resistant marker
genes may be used in the two homologous recombinations.
[0068] Furthermore, the present invention provides a method of
using the pluripotent cell as a recipient cell into which a foreign
gene(s) or a fragment(s) thereof, or a foreign chromosome(s) or a
fragment(s) thereof are to be transferred. The foreign gene(s) or
fragment(s) thereof may be incorporated in a vector such as a
plasmid, a cosmid, YAC or the like. Alternatively, the foreign
chromosome(s) or fragment(s) thereof may be contained in a
microcell. The foreign chromosome(s) or fragment(s) thereof is
preferably, but not limited to, one that contains a gene(s)
identical with or homologous to the endogenous gene(s) disrupted in
the pluripotent cell. The term "homologous gene" means herein a
gene encoding the same kind of protein or a protein having a
similar property in the same or different species of a given
organism.
[0069] Moreover, the present invention provides a method of using
the pluripotent cell for producing a chimeric non-human animal.
[0070] The present invention also provides a method of producing a
pluripotent cell containing a foreign chromosome(s) or a
fragment(s) thereof, which comprises the steps of:
[0071] preparing a microcell containing the foreign chromosome(s)
or fragment(s) thereof; and
[0072] fusing the microcell with said pluripotent cell having at
least two endogenous genes disrupted, whereby said foreign
chromosome(s) or fragment(s) thereof is transferred into said
pluripotent cell.
[0073] The present invention further provides a method of producing
a chimeric non-human animal, which comprises the steps of:
[0074] preparing a microcell containing a foreign chromosome(s) or
a fragment(s) thereof; and
[0075] fusing the microcell with said pluripotent cell having at
least two endogenous genes disrupted, whereby said foreign
chromosome(s) or fragment(s) thereof is transferred into said
pluripotent cell.
[0076] In the aforementioned two methods, the foreign chromosome(s)
or fragment(s) thereof may have a length(s) of at least 1 Mb (100
million base pairs). The foreign chromosome(s) or a fragment(s)
thereof may contain a region encoding an antibody. The microcell
containing the foreign chromosome(s) or fragment(s) thereof may be
induced from a hybrid cell prepared by the fusion of a cell
containing the foreign chromosome(s) or fragment(s) thereof, with a
cell having a high ability to form a microcell. The microcell
containing the foreign chromosome(s) or fragment(s) thereof may be
induced from a cell prepared by a further fusion of the microcell
induced from the hybrid cell, with a cell having a high ability to
form a microcell. The cell containing the foreign chromosome(s) or
fragment(s) thereof may be a human normal diploid cell. The cell
having a high ability to form a microcell may be a mouse A9 cell.
In the methods of producing a chimeric non-human animal, a foreign
chromosome(s) or a fragment(s) thereof containing gene(s) identical
with or homologous to the endogenous gene(s) disrupted in the
pluripotent cell may be transferred into the pluripotent cell
having the disrupted at least two endogenous genes and then, a
chimera of the cell with an embryo of a non-human animal in a
strain deficient in a gene(s) identical with or homologous to said
endogenous gene(s) may be prepared. The chimeric non-human animal
deficient in a gene identical with or homologous to the endogenous
gene disrupted in said pluripotent cell may be produced by
homologous recombination in gene targeting. The chimeric non-human
animal may be such that it retains the foreign chromosome(s) or
fragment(s) thereof, expresses a gene(s) on the foreign
chromosome(s) or fragment(s) thereof, and can transmit the foreign
chromosome(s) or fragment(s) thereof to its progeny. The chimeric
non-human animal may be a mammal, preferably a mouse.
[0077] The present invention also provides a pluripotent cell
containing a foreign chromosome(s) or a fragment(s) thereof, which
is obtainable by a method of producing a chimeric non-human animal,
which method comprises the steps of:
[0078] preparing a microcell containing the foreign chromosome(s)
or fragment(s) thereof; and
[0079] fusing the microcell with said pluripotent cell having at
least two endogenous genes disrupted, whereby said foreign
chromosome(s) or fragment(s) thereof is transferred into said
pluripotent cell. The present invention further provides a method
of using the cell for producing a chimeric non-human animal.
[0080] The present invention also provides a chimeric non-human
animal retaining a foreign chromosome(s) or a fragment(s) thereof
and expressing the gene(s) on the foreign chromosome(s) or
fragment(s) thereof, which is obtainable by one of the
aforementioned methods of producing a chimeric non-human animal, or
its progeny. The present invention also provides a non-human animal
retaining a foreign chromosome(s) or a fragment(s) thereof and
expressing the gene(s) on the foreign chromosome(s) or fragment(s)
thereof which is obtainable by mating between the chimeric
non-human animals or its progenies, or its progeny. The present
invention further provides a tissue from the aforementioned
chimeric non-human animal or its progeny, or the aforementioned
non-human animal or its progeny. The present invention still more
provides a cell from the aforementioned chimeric non-human animal
or its progeny, or the aforementioned non-human animal or its
progeny. The cell may be a B cell.
[0081] The present invention also provides a hybridoma prepared by
the fusion of the cell from the aforementioned chimeric non-human
animal or its progeny, or the aforementioned non-human animal or
its progeny with a myeloma cell.
[0082] The present invention provides a non-human animal or its
progeny retaining a foreign chromosome(s) or a fragment(s) thereof
and expressing a gene(s) on the foreign chromosome(s) or
fragment(s) thereof, which is obtainable by mating said chimeric
non-human animal or its progeny or said non-human animal or its
progeny retaining the foreign chromosome(s) or fragment(s) thereof
and expressing the gene(s) on the foreign chromosome(s) or
fragment(s) thereof, with a non-human animal in a stain deficient
in a gene(s) identical with or homologous to said gene(s).
[0083] Furthermore, the present invention provides a method of
producing a biologically active substance, which comprises
expressing a gene(s) on a foreign chromosome(s) or a fragment in
the chimeric non-human animal or its progeny, or the non-human
animal or its progeny, or a tissue or a cell thereof and recovering
the biologically active substance as the expression product. The
cell of the chimeric non-human animal or its progeny, or the
non-human animal or its progeny may be a B cell. The B cell may be
immortalized by fusion with a myeloma cell. The biologically active
substance may be an antibody. The antibody may be an antibody of
mammal, preferably a human antibody.
[0084] Moreover, the present invention provides a method of
producing a biologically active substance, which comprises
expressing a gene(s) on a foreign chromosome(s) or a fragment in a
offspring or a tissue and a cell thereof, wherein the offspring is
produced by mating the chimeric non-human animal or its progeny, or
the non-human animal or its progeny retaining the foreign
chromosome(s) or fragment(s) thereof with a non-human animal in a
strain deficient in a gene identical with or homologous to said
genes, and expressing the gene(s) on the foreign chromosome(s) or
fragment(s) thereof, and recovering the biologically active
substance as the expression product.
[0085] The present invention also provides a vector containing a
foreign chromosomal gene(s) for use in gene transfer into a
non-human animal and a non-human animal cell. The foreign
chromosome(s) is preferably one from human, more preferably a human
chromosome #14 fragment. The non-human animal is preferably a
mouse.
[0086] The term "allele" is used herein.
[0087] The term "homologous gene" means herein a gene encoding the
same kind of protein or a protein having a similar property in the
same or different species of a given organism.
[0088] According to the present invention, a chimeric non-human
animal retaining a foreign chromosome(s) or a fragment(s) thereof
and expressing the gene(s) on the chromosome(s) or fragment(s) is
provided. The chimeric non-human animal of the present invention
can be used to produce biologically active substances.
[0089] According to the present invention, a pluripotent cell
retaining a foreign chromosome(s) or a fragment(s) thereof and
expressing a gene(s) on the chromosome(s) or fragment(s) thereof is
provided. The pluripotent cell can be used for treatment of
hereditary diseases, for example, by bone marrow
transplantation.
[0090] According to the present invention, a pluripotent cell
having at least two endogenous genes disrupted is provided. The
cell of the present invention can be used as a recipient cell for
transferring a foreign chromosome(s) or a fragment(s) thereof
containing a gene identical with or homologous to the disrupted
endogenous genes to produce a functional cell or a chimeric
non-human animal retaining the foreign chromosome(s) or fragment(s)
thereof and expressing the gene(s) on the chromosome(s) or
fragment(s). A biologically active substance(s) can be produced as
a gene product(s) by expressing the gene(s) on the chromosome(s) or
fragment(s) thereof in the chimeric non-human animal or its
progeny, or a tissue or a cell thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0091] FIG. 1 shows the results of PCR analysis of an A9 cell
retaining human chromosome #2 (fragment).
[0092] FIG. 2 shows that human chromosome #22 (fragment) is
retained in an E14 drug resistant cell (PCR analysis).
[0093] FIG. 3 is a photograph of electrophoresis patterns showing
that human L1 sequence is retained in a chimeric mouse produced
from a human chromosome #22-transferred ES cell (Southern
analysis).
[0094] FIG. 4 is a photograph of electrophoresis patterns showing
the presence of a human chromosome in organs of a human chromosome
#22 transferred chimeric mouse (PCR analysis).
[0095] FIG. 5 is a photograph of electrophoresis patterns showing
the results of the expression of human genes in a human chromosome
#22 transferred chimeric mouse (RT-PCR).
[0096] FIG. 6 is a photograph of electrophoresis patterns showing
the results of the expression of human genes in organs of a human
chromosome #22 transferred chimeric mouse (RT-PCR).
[0097] FIG. 7 shows that human chromosome #4 (fragment) is retained
in an E14 drug resistant cell (PCR analysis).
[0098] FIG. 8 is a photograph of electrophoresis patterns showing
the detection of human L1 sequence in a human chromosome
#4-transferred E14 cell clone (Southern analysis).
[0099] FIG. 9 is a photograph of electrophoresis patterns showing
that human L1 sequence is retained in a chimeric mouse produced
from a human chromosome #4-transferred ES cell (Southern
analysis).
[0100] FIG. 10 shows that human chromosome #14 (fragment) is
retained in a TT2 drug resistant cell (PCR analysis).
[0101] FIG. 11 is a photograph of electrophoresis patterns showing
the presence of a human chromosome in organs of a chimeric mouse
produced from a human chromosome #14 transferred ES cell (PCR
analysis).
[0102] FIG. 12 shows the results of a test on a tail-derived
fibroblast cell for resistance to G418.
[0103] FIG. 13 shows the concentration of human antibody IgM in a
serum of a human serum albumin (hereinafter referred to as
"HSA")-immunized chimeric mouse (ELISA).
[0104] FIG. 14 shows the concentration of human antibody IgG in a
serum of an HSA-immunized chimeric mouse (ELISA).
[0105] FIG. 15 shows the results of ELISA of hybridoma clone H4B7
capable of producing human IgM.
[0106] FIG. 16 is a photograph of the results of FISH analysis of a
mouse ES cell clone (TT2 cell clone PG15) retaining partial
fragments of human chromosomes #2 and 14.
[0107] FIG. 17 shows that the antibody titer of anti-HSA human IgG
is increased in a serum of an HSA-immunized chimeric mouse.
[0108] FIG. 18 shows that the antibody titer of anti-HSA human
Ig.kappa. is increased in a serum of an HSA-immunized chimeric
mouse.
[0109] FIG. 19 is a photograph of electrophoresis patterns showing
the detection of human L1 sequence in a human chromosome
#22-transferred TT2 cell clone (Southern analysis).
[0110] FIG. 20 shows that the antibody titer of anti-HSA human
Ig.lambda. is increased in a serum of an HSA-immunized chimeric
mouse.
[0111] FIG. 21 shows that a partial fragment of human chromosome #2
is retained in a progeny of a chimeric mouse into which a partial
fragment of a human chromosome #2 was transferred (PCR
analysis).
[0112] FIG. 22 shows the presence of a cell expressing human .mu.
chain on the cell surface in a spleen of a human chromosome
#14-transferred chimeric mouse (flow cytometry analysis).
[0113] FIG. 23 shows the structure of LoxP-pstNEO plasmid DNA.
[0114] FIG. 24 shows the structure of genomic DNA carrying a mouse
antibody heavy chain C.mu. gene.
[0115] FIG. 25 shows the structure of genomic DNA carrying a mouse
antibody light-chain .kappa. gene.
[0116] FIG. 26 shows the structures of a mouse antibody heavy-chain
targeting vector and a probe for Southern blotting, as well as a
DNA fragment to be detected in homologous recombinants.
[0117] FIG. 27 shows the structures of a mouse antibody light-chain
.kappa. targeting vector and a probe for Southern blotting, as well
as a DNA fragment to be detected in homologous recombinants.
[0118] FIG. 28 is a photograph of electrophoresis patterns showing
the results of Southern blot analysis of mouse antibody heavy-chain
homologous recombinants and high concentration G418 resistant
clones derived therefrom.
[0119] FIG. 29 shows a photograph of electrophoresis patterns
showing the results of Southern blot analysis of mouse antibody
light-chain homologous recombinants.
[0120] FIG. 30 shows the structure of pLoxP-PGKPuro plasmid
DNA.
[0121] FIG. 31 shows a mouse antibody light-chain .kappa. targeting
vector, a probe for use in the southern blot analysis of genomic
DNA from transformant TT2F cells, and DNA fragments to be detected
in homologous recombinants.
[0122] FIG. 32 shows a photograph of electrophoresis patterns
showing the results of Southern blot analysis of high concentration
G418 resistant cell clones derived from mouse antibody light-chain
homologous recombinants.
[0123] FIG. 33 shows that the antibody titers of anti-HSA human IgH
antibodies are increased in a serum of an HSA-immunized chimeric
mouse.
[0124] FIG. 34 is a photograph of the result of FISH analysis of an
antibody heavy- and light-chains deficient mouse ES cell clone
retaining partial fragments of human chromosomes #2 and #14.
[0125] FIG. 35 shows that the antibody titers of anti-HSA human Ig
antibodies are increased in a serum of an HSA-immunized chimeric
mouse.
[0126] FIG. 36 shows a photograph of the result of FISH analysis of
a mouse A9 cell containing human chromosome #14 (human centromere
sequence probe).
[0127] FIG. 37 shows a photograph of the result of FISH analysis of
a mouse A9 cell containing human chromosome #14 (human
chromosome-specific probe).
[0128] FIG. 38 shows the results of a test for stability of human
chromosome fragments (#14: SC20, #2:W23) in a mouse ES cell.
[0129] FIG. 39 shows the results of analysis for stability of human
chromosome #14 fragments in a mouse.
[0130] FIG. 40 shows the results of PCR analysis of a G418
resistant hybrid cells retaining human chromosome #22
(fragment).
[0131] FIG. 41 shows the results of FISH analysis of an A9 cell
retaining fragmented human chromosome #22.
[0132] FIG. 42 shows the results of complete human
antibody-producing mouse strains established by mating.
[0133] FIG. 43 shows the results of the determination of the
concentration of human antibody .kappa. chain in a serum of a mouse
retaining a human chromosome #2 fragment, W23.
[0134] FIG. 44 shows the results of the determination of the
concentration of human antibody .kappa. and .lambda. chains in a
serum of a mouse.
[0135] FIG. 45 shows the structure of pBS-TEL/LIFPuro.
[0136] FIG. 46 shows that human chromosome #22 is retained in a
DT40 cell clone.
[0137] FIG. 47 shows the identification of homologous recombinant
in LIF locus.
[0138] FIG. 48 shows the fragmentation of human chromosome #22 in a
DT40/#22neo cell clone.
[0139] FIG. 49 shows a DT40 cell clone retaining full length or
fragmented human #22 chromosome.
BEST MODE FOR CARRYING OUT THE INVENTION
[0140] The present invention will now be described in detail.
[0141] A non-human animal that retains a human chromosome(s) or a
fragment(s) thereof and which expresses the gene on the
chromosome(s) or fragment(s) thereof can be produced by
[0142] (1) preparing a chromosome donor cell which retains a
labeled human chromosome or a fragment thereof;
[0143] (2) transferring the human chromosome or fragment thereof
into a non-human animal pluripotent cell by microcell fusion;
[0144] (3) producing a chimeric non-human animal from the cell;
and
[0145] (4) confirming that the human chromosome is retained in the
chimeric non-human animal and that a human gene is expressed.
[0146] In this procedure, a mouse is used as a non-human animal
that retains a human chromosome or a fragment thereof and which
expresses the gene on the chromosome or fragment thereof (the mouse
is hereinafter referred to as a "human chromosome transferred
mouse").
[0147] The term "human chromosome" means a naturally occurring
complex which consists of nucleic acids and proteins that are
derived from human cells. There are 46 normal human chromosomes of
23 kinds (24 kinds in male), each of which contains DNAs of about
50-300 Mb in size. In the present invention, the human chromosome
includes not only partial fragments which can be stably replicated
and segregated as independent chromosomes but also fragments that
are translocated on mouse chromosomes and which are retained
stably. The size of the DNA is usually at least 1 Mb and in some
cases, it is smaller than 1 Mb. The feature of the present
invention resides in that a mouse can retain and express the
foreign gene on a foreign chromosome as a mediator without
treatments such as cloning in an E. coli or yeast cell, or
extraction of the DNA from a cell.
[0148] The term "human chromosome transferred mouse" means a mouse
retaining a human chromosome(s) or a fragment(s) thereof in all or
part of its normal somatic cells. The mouse expresses the gene(s)
on a human chromosome(s) or a fragment(s) thereof in all or part of
its normal somatic cells.
[0149] (1) Preparation of a Chromosome Donor Cell Which Retains a
Labeled Human Chromosome or a Fragment Thereof
[0150] A desired chromosome donor cell 1) retains a human
chromosome(s) labeled with a marker also available for selection of
recipient cells; 2) does not contain other human chromosomes; and
3) has a higher ability to form a microcell.
[0151] Any human-derived cell lines, cancer cells and primary
culture cells can be used as materials for providing human
chromosomes. Among them, normal fibroblast cells are suitable
because they have a low possibility of abnormality such as deletion
and amplification of chromosomes and can be readily cultured.
[0152] As for 1), human cells can be transformed with vectors that
express genes for markers such as drug-resistance (e.g., G418-,
puromycin-, hygromycin- or blasticidin-resistance). Promoters
operating efficiently not only in human cells but also in recipient
cells such as mouse ES cells are desirably used to regulate the
expression of the marker used. For this purpose, herpes simplex
virus thymidine kinase promoter linked with SV 40 enhancer (Katoh
et al., Cell Struct. Funct., 12:575, 1987), mouse PGK-1 promoter
(Soriano et al., Cell, 64:693, 1991) and the like can be used. A
library of human cell transformants in which the introduced marker
genes have been inserted into 46 human chromosomes of 23 kinds at
random can be prepared by transformation through electroporation
(Ishida et al., "Cell Technology Experiment Manual", published by
Kodansha, 1992) and the like and subsequent selection of
transformants.
[0153] As for 3), since many human normal cells have a very low
ability to form microcells, the whole cell of the transformant may
be fused with a cell having a high ability to form microcells such
as mouse A9 cell (Oshimura, M., Environ. Health Perspect., 93:57,
1991) so as to provide the transformed cell with an ability to form
microcells. It is known that in mouse-human hybrid cells, human
chromosomes selectively disappear. The fused cell selected by the
marker can retain stably the marked human chromosome.
[0154] In order to meet the condition of 2), it is desired to
obtain a microcell from the fused cell and fuse it again with a
mouse A9 cell. In this case, too, most of the cells selected by the
marker will meet the three conditions 1), 2) and 3) above. The
marked human chromosomes can be identified in the finally obtained
mouse-human monochromosomal hybrid cells by PCR (Polymerase Chain
Reaction, Saiki et al., Science, 239:487, 1988), Southern blot
analysis (Ausubel et al., Current protocols in molecular biology,
John Wiley & Sons, Inc., 1994), FISH analysis (Fluorescence In
situ Hybridization, Lawrence et al., Cell, 52: 51, 1988) and the
like. If the transfer of a specified chromosome is desired, the
above procedures are applied to each of many human cell
transformant clones to select a clone in which a chromosome of
interest is marked. Alternatively, the above procedures are applied
to a mixture of human cell transformant clones and the
identification of human chromosomes is carried out on a large
number of the resulting mouse-human monochromosome hybrid
cells.
[0155] In addition, a marker gene can be inserted into a desired
site by homologous recombination of a specific DNA sequence on the
chromosome which is to be transferred (Thomas et al., Cell, 51:503,
1987).
[0156] A microcell prepared from the mouse-human hybrid cell may be
irradiated with .gamma.-rays such that the marked human chromosome
is fragmented and transferred into a mouse A9 cell. Even if the
microcell is not irradiated with .gamma.-rays, a partially
fragmented human chromosome may be transferred at a certain
frequency. In these cases, the resulting microcell fused clones
retain partial fragments of the marked human chromosomes. These
clones can be used when it is desired to transfer the partial
fragments into recipient cells.
[0157] Human chromosomes to be introduced into ES cells may be
modified by deletion, translocation, substitution and the like.
Specific procedures for these modifications are as follows:
[0158] 1) In each of the steps of preparing the aforementioned
mouse-human hybrid cell, inducing a microcell from the mouse-human
hybrid cell, further fusing the microcell with a mouse A9 cell,
inducing a microcell from the further fused cell and fusing the
latter microcell with a mouse ES cell, deletion and/or
translocation of human chromosomes may occasionally occur. Cells
retaining such mutated chromosomes are selected under the
microscopic observation of chromosomes or by use of PCR, Southern
analysis, or the like. A clone retaining a desired mutant
chromosome can be selected from a mouse A9 library retaining
various human chomosomes. A clone retaining a desired mutant
chromosome can be selected from A9 or ES cell fused with a
microcell induced from a mouse A9 cell retaining a certain human
chromosome. The frequency of fragmentation of chromosomes can be
raised by .gamma.-ray irradiation (Koi et al., Science, 260:361,
1993).
[0159] 2) A targeting vector retaining a loxp sequence that is
recognized by Cre enzyme is constructed. A clone into which a loxp
sequence has been inserted at a desired site on a chromosome is
obtained by homologous recombination in a cell retaining a human
chromosome. Subsequently, Cre enzyme is expressed in the cell of
the clone to select a mutant having chromosomal deletion and/or
translocation caused by site-specific recombination. See WO97/49804
and Smith et al., Nature Genetics, 9:376, 195. As a host into which
a targeting vector is to be introduced, a cell allowing for
high-frequency homologous recombination such as DT40 cell (Dieken
et al., Nature Genetics, 12:174, 1996) may also be used.
[0160] 3) A targeting vector retaining a human telomere sequence is
constructed and the telomere sequence is inserted in the cell at a
desired site on a chromosome by homologous recombination in a cell
retaining a human chromosome. After a clone into which the telomere
sequence has been inserted is obtained, a mutant having deletion
caused by the telomere truncation is obtained. See Itzhaki et al.,
Nature Genet., 2, 283-287, 1992 and Brown et al., P. N. A. S.,
93:7125, 1996. As a host into which a targeting vector is to be
introduced, a cell allowing for high-frequency homologous
recombination such as DT40 cell (Dieken et al., supra) may also be
used. Telomere truncation of human chromosomes in DT40 cell is
first disclosed in the present invention. Brown (supra) discloses
that a vector was inserted into a repeat sequence on a chromosome.
However, no specific site can be targeted. Itzhaki et al. discloses
that tumor cells, i.e., 12000 cells of cell line HT1080 into which
a telomere sequence was introduced were analyzed and 8 homologous
recombinants were obtained. They found that out of the 8 cells,
only one caused deletion by insertion of the telomere sequence. For
some kinds of cells, results were reported that no mutant having
truncation was obtained by insertion of a telomere sequence into
some kinds of cells (Barnett et al., Nucleic Acids Res., 21:27,
1993). In spite of this report, the inventors believed that it was
necessary to increase the absolute number of homologous
recombinants in order to obtain mutants having truncation and made
an attempt to perform telomere truncation using a DT40 cell as a
host. As a result, it was surprisingly found that truncation
occurred in all of the 8 homologous recombinants obtained.
[0161] As mentioned above, a gene that should not be expressed in a
human chromosome-transferred mouse can be removed by modification
of a introduced chromosome. If the size of a chromosome to be
transferred is shortened by fragmentation, the chromosome fragment
to be transferred can be transmitted to progenies of the
chromosome-transferred mice. In addition, using chromosome
translocation and substitution techniques, genes derived from a
plurality of chromosomes can be expressed on the same chromosome
fragment and portions of a plurality of genes on the chromosome
fragments can be replaced with different genes. In other words,
foreign chromosome fragments can be used as vectors for
transferring genes into individual mice and their cells.
[0162] (2) Transfer of the Human Chromosome or Fragment Thereof
into a Mouse Pluripotent Cell
[0163] It has been reported to date that an embyonic carcinoma cell
(EC cell, Hanaoka et al., Differentiation, 48:83, 1991), an
embyonic stem cell (ES cell, Evans, Nature, 292:154, 1981) or an
embyonic germ cell (EG cell, Matsui et al., Cell, 70:841, 1992)
that are derived from various strains of mice contribute to the
normal somatic cells in mice, or are capable of the production of
chimeric mice, by injection into or coculturing with a mouse early
embryo. ES and EG cells have a very high ability in this respect
and in many cases, they also contribute to germ cells thereby
making it possible to produce progenies derived from the cells. EC
cells can be obtained predominantly from teratocarcinoma; ES cells
from the inner cell masses of blastocysts; and EG cells from
primordial germ cells appearing at the early stage of embryogeny.
These cell lines and their mutants, and any undifferentiated cells
that are capable of differentiation into all or part of the normal
somatic cells in mice can be used as recipient cells for the
transfer of human chromosome in the present invention. In these
recipient cells, for the purpose of achieving advantageous
expression of a human gene to be introduced, a gene or genes such
as a mouse gene homologous to the human gene can be disrupted in a
chimeric mouse or a chimeric-mouse derived tissue or cell by using
homologous recombination in gene targeting (Joyner et al., Gene
Targeting, 1993, IRL PRESS) or other techniques.
[0164] The microcells prepared from the human chromosome donor
cells or the microcells irradiated with .gamma.-rays can be used as
materials for the transfer of human chromosomes into the recipient
cells. The human chromosome can be transferred into the recipient
cell through fusion of the recipient cell with the microcell by the
method described in Motoyuki Shimizu, "Cell Technology Handbook",
published by Yodosha, 1992. The microcell donor cells retain
markers by which human chromosomes or fragments thereof can be
selected in the recipient cells. The clone containing a gene, a
chromosome or a fragment of interest can be selected by PCR,
Southern blot analysis, FISH method or the like in the same manner
as in (1), thus all kinds of human chromosomes or fragments thereof
can be transferred. Moreover, if several chromosomes or fragments
thereof which contain different selection markers are transferred
sequentially, a recipient cell retaining these chromosomes or
fragments at the same time can be obtained. In addition, clones
having an increased number of the transferred chromosome can be
selected from the clones into which the human chromosome has been
transferred. Such selection can be accomplished by increasing the
concentration of a selection drug to be added to a culture
medium.
[0165] In order to determine whether the recipient cell selected by
the marker (e.g., G418 resistance) on the human chromosome retains
the whole or part of the chromosome retained by the donor cell, the
following confirmative techniques may be employed: Southern blot
analysis using the genomic DNA extracted from the selected
recipient cell, with a human specific repeated sequence (L1, Alu,
etc.: Korenberg et al., Cell, 53:391, 1988) or a human gene used as
a probe; and chromosome analysis such as PCR method using a human
gene specific primer or FISH method using a human chromosome
specific probe (Lichter et al., Human Genetics, 80:224, 1988).
[0166] (3) Production of a Chimeric Mouse from the Human Chromosome
Transferred ES Cell
[0167] The method described in Shinichi Aizawa, "Biotechnology
Manual Series 8, Gene Targeting", published by Yodosha, 1995 may be
used to produce chimeric mice from the ES cell clone obtained in
(2). In selecting factors for efficient production of chimeric
mice, such as the developmental stage of the host embryo and its
strain, it is desired to employ the conditions already reviewed for
the respective ES cell clones. For example, 8 cell stage embryos
derived from Balb/c (albino, CREA JAPAN, INC.) or ICR (albino, CREA
JAPAN, INC.) are desirably used for CBA.times.C57BL/6 F1-derived
TT2 cell (agouti, Yagi et al., Analytical Biochemistry, 214:70,
1993).
[0168] (4) Confirmation of the Retention of the Human Chromosome in
the Chimeric Mice and the Expression of a Human Gene
[0169] The contribution of the ES cells in mice produced from the
embryos into which ES cells were injected can be roughly judged by
the color of their coat. However, it should be noted that the total
absence of contribution to the coat color does not always lead to
the conclusion that there is no contribution to other tissues. The
detailed information on the retention of the human chromosome in
various tissues of the chimeric mice can be obtained by Southern
blot analysis using the genomic DNA extracted from various tissues,
by PCR or the like.
[0170] The expression of the gene on the transferred human
chromosome can be confirmed by the following methods. The
expression of mRNA transcribed from the human chromosome can be
detected by RT-PCR method or northern blotting (Ausubel et al.,
supra) using RNAs derived from various tissues (Kawasaki et al.,
P.N.A.S., 85:5698, 1988). The expression at the protein level can
be detected by enzyme immunoassay using an anti-human protein
antibody that is rendered minimal in its ability to enter into a
cross reaction with mouse homologous proteins (ELISA, Toyama and
Ando, "Monoclonal Antibody Experiment Manual", published by
Kodansha Scientific, 1987; Ishikawa, "Enzyme immunoassay with
Superhigh Sensitivity", published by Gakkai Shuppan Center, 1993),
western blotting (Ausuel et al., supra), isozyme analysis utilizing
the difference in electrophoretic mobility (Koi et al., Jpn. J.
Cancer Res., 80:413, 1989) or the like. The retention of the human
chromosome in the chimeric mice and the expression of the gene on
the human chromosome can be confirmed by the appearance of the
cells expressing a drug resistance marker gene in primary culture
cells derived from the chimeric mice.
[0171] For example, human IgM, IgG, IgA and the like in sera of the
chimeric mice which are produced from ES cells retaining human
chromosome #14 on which a gene for human immunoglobulin heavy chain
exists can be detected by enzyme immunoassay using an anti-human Ig
antibody that is rendered minimal in its ability to enter into
cross reaction with mouse antibody. Hybridomas capable of producing
a human immonoglobulin heavy chain can be obtained by ELISA
screening of hybridomas prepared by immunizing the chimeric mouse
with a human-derived antigen (e.g., HSA) and fusing the spleen
cells of the immunized mice with mouse myeloma cells (Toyama and
Ando, "Monoclonal Antibody Experiment Manual", published by
Kodansha Scientific, 1987).
[0172] The method for producing a chimeric non-human animal of the
present invention has been explained above with reference to the
case of a mouse retaining a human chromosome(s) or a fragment(s)
thereof and expressing the gene(s) on the chromosome(s) or
fragment(s). In the present invention, chromosomes or fragments
thereof to be transferred into chimeric non-human animals are not
limited to those derived from humans but include any foreign
chromosomes and fragments thereof. The term "foreign chromosome"
means a chromosome which is transferred into a pluripotent cell
and, subsequently, the gene on which (or a fragment thereof) is
expressed in a chimeric non-human animal. The organism species from
which the foreign chromosome is derived is not particularly
limited. Other kinds of chimeric animals such as chimeric rat and
pig can be produced by the method of the present invention. ES
cells or ES-like cells derived from animals other than mouse were
established with rat (Iannaccone et al., Dev. Biol., 163, 288-,
1994), pig (Wheeler et al., Reprod. Fertil. Dev., 6, 563-, 1994)
and bovine (Sims et al., Proc. Natl. Acad. Sci. USA, 91, 6143-6147,
1994) and attempts have been made on cyprinodont, chicken and the
like ("Transgenic Animal", Protein.cndot.Nucleic Acid.cndot.Enzyme,
October, 1995, Special Issue, published by Kyoritsu Shuppan). It is
known that sheep is developed normally from an unfertilized egg
transplanted with the nucleus from ES-like cell (ED cell) or
epithelial-like cell obtained by subcultivation of the ES-like cell
through at least 10 generations (Campbell et al., Nature, 380, 64-,
1996). These ES cells and ES-like cells can be used as recipient
cells in the transfer of foreign chromosomes to produce chimeric
non-human animals retaining the foreign chromosomes or fragments
thereof and expressing the genes on the chromosomes or fragments
thereof in the same manner as in the case of mouse.
[0173] In the present invention, pluripotent cells into which a
foreign chromosome(s) or a fragment(s) thereof are transferred are
not limited to the ES cells, EC cells and EG cells mentioned above.
For example, it is possible to transfer a foreign chromosome(s) or
a fragment(s) thereof into bone marrow stem cells. If these bone
marrow stem cells are transplanted into a living organism,
hereditary diseases, etc. may be treated.
[0174] If an ES cell retaining a foreign chromosome(s) or a
fragment(s) thereof is differentiated to a germ cell in the
chimeric non-human animal, reproduced progenies will retain the
transferred chromosome(s) or fragment(s) thereof and express the
gene(s) on the chromosome(s) or fragment(s) thereof.
[0175] The chimeric non-human animals or their progenies can be
used to express the gene on the foreign chromosome or fragment
thereof and to recover the expression product, thereby producing a
biologically active substance. More specifically, the chimeric
non-human animals or their progenies can be bred under the
conditions for expressing the gene on the foreign chromosome or
fragment thereof to recover the expression product from the blood,
ascites and the like of the animals. Alternatively, the tissues or
cells of the chimeric non-human animal, or immortalized cells
derived therefrom (e.g., hybridomas immortalized by fusion with
myeloma cells) can be cultured under the conditions for expressing
the gene on the foreign chromosome or fragment thereof and the
expression product is thereafter recovered from the culture.
Furthermore, a foreign chromosome(s) or a fragment(s) thereof which
was extracted from tissues or cells of these chimeric non-human
animals or their progenies, or from immortalized cells derived
therefrom; the DNA which is a component of said foreign
chromosome(s) or fragment(s) thereof; or cDNA derived from the
foreign chromosome(s) or fragment(s) thereof retained in tissues or
cells of the chimeric non-human animals or their progenies, or in
immortalized cells derived therefrom may be used to transform
animal cells or insect cells (e.g., CHO cells, BHK cells, hepatoma
cells, myeloma cells, SF9 cells) and the transformed cells may be
cultured under the conditions for expressing the gene on the
foreign chromosome(s) or fragment(s) thereof to recover the
expression product (e.g., an antibody protein specific to a
particular antigen) from the culture. The expression product can be
collected by known techniques such as centrifugation and purified
by known techniques such as ammonium sulfate fractionation,
partition chromatography, gel filtration chromatography, adsorption
chromatography, preparative thin-layer chromatography and the like.
The biologically active substance includes any kinds of substances
encoded on foreign chromosomes, for example, antibodies,
particularly human antibodies. For example, the human antibody gene
on the foreign chromosome can be cloned from spleen cells of the
chimeric non-human animal or immortalized cells such as hybridomas
derived therefrom and transferred into Chinese hamster ovary cells
(CHO), myeloma cells or the like to produce a human antibody
(Lynette et al., Biotechnology, 10:1121-, 1992; Bebbington et al.,
Biotechnology, 10:169-, 1992).
[0176] The chimeric mice or their progenies that retain human
chromosomes #2, 14 and/or 22 (or fragments thereof) which can be
produced by the method of the present invention can retain the
greater part of the functional sequences of respective genes for
human antibody heavy chain on chromosome #14, light chain.lambda.
on chromosome #2 and light chain .lambda. on chromosome #22. Hence,
they can produce a wide repertory of antibodies which are more
similar to human antibody repertory, compared with known transgenic
mice into which parts of human antibody gene have been transferred
by using yeast artificial chromosomes and the like (Green et al.,
Nature Genetics, 7, 13-, 1994; Lonberg et al., Nature, 368, 856-,
1994). Also, the chimeric mice and their progenies retaining two
human chromosomes (or fragments) of #2+#14, #22+#14 or other
combination and the mice and their progenies retaining three human
chromosomes (or fragments) of #2+#14+#22 or other combination which
are obtainable by mating said chimeric mice and their progenies
retaining two human chromosomes (or fragments), as produced by the
method of the invention, can produce complete human antibodies both
heavy- and light-chains of which are derived from human. These mice
can recognize human-derived antigens as foreign substances to cause
an immunoreaction with the antigens, thereby producing
antigen-specific human antibodies. These properties can be utilized
to produce human monoclonal and polyclonal antibodies for
therapeutic treatments (Green et al, supra; Longberg et al.,
supra). On the other hand, in order to obtain a human antibody
having high affinity for a particular antigen more efficiently, it
is desirable to produce a mouse which produces a human antibody but
not a mouse antibody (Green et al., supra; Lonberg et al., supra).
In the present invention, this is achieved typically by the
following Method A or B using known techniques.
[0177] Method A: a method using a mouse antibody-deficient ES cell
and a mouse antibody-deficient host embryo for chimera
production.
[0178] Method B: a method in which a progeny retaining a human
chromosome is obtained from a human chromosome-transferred chimeric
mouse, followed by mating said progeny with a mouse in a strain
deficient in a mouse antibody gene.
[0179] A typical example for each of Methods A and B will be
described below specifically.
[0180] Specific Procedures for Method A
[0181] 1. One allele of a mouse antibody heavy-chain gene present
in two copies in a mouse ES cell is disrupted by homologous
recombination in gene targeting (Joyner et al., "Gene Targeting",
published by IRL PRESS, 1993). A marker gene, such as a G 418
resistance gene, sandwiched with two copies of a sequence which can
be removed later by site-specific recombination [for example,
lo.times.P sequence (see recombination with Cre recombinase in
Sauer et al., supra; and see also the use of FLP recombinase-FRT
sequence in O'Gorman, Science, 251;1351-, 1991)] is inserted at the
site where the targeted gene is disrupted.
[0182] 2. The resultant drug-resistant mouse ES cells in which one
allele of an antibody heavy-chain gene was disrupted is cultured in
the presence of the drug at a high concentration. Then, those
clones which became high concentration drug-resistant are selected.
By screening these clones, clones in which both antibody
heavy-chain genes were disrupted can be obtained (Shinichi Aizawa,
supra).
[0183] Alternatively, the other allele of a target gene in the
drug-resistant mouse ES cell in which one allele of the antibody
heavy-chain gene has been disrupted is also disrupted by homologous
recombination. The same procedure may be repeated using a marker
gene other than the precedingly inserted marker gene. For example,
homologous recombination is performed using a G418-resistance gene,
followed by another homologous recombination using a
puromycin-resistance gene to obtain clones in which both alleles of
the antibody heavy-chain gene have been disrupted. When the same
marker as the precedingly inserted marker is used, an enzyme gene
that can cause site-specific recombination between recombinant
sequences inserted at the both ends of the drug-resistance gene of
item 1 is transiently introduced. Subsequently, drug-sensitive
clones are selected that are free of the drug-resistance gene that
has been inserted in the target gene. Then, a marker gene is
inserted again by homologous recombination in gene targeting to
obtain clones in which both alleles of the target gene have been
disrupted (Seishi Takatsu et al., Experimental Medicine,
supplement, Basic Techniques for Immunological Study, p. 255-,
1995, Yodosha).
[0184] 3. An enzyme gene (e.g., a Cre recombinase gene (Sauer et
al., supra)) which causes a site-specific recombination between the
recombination sequences inserted at both the ends of the
drug-resistance gene in step 1 above is transiently transferred
into the mouse ES cells from step 2 above in which both antibody
heavy-chain genes were disrupted. Then, drug-sensitive clones are
selected in which the drug-resistance genes inserted at the sites
of both heavy-chain genes were deleted as a result of recombination
between the loxP sequences [Seiji Takatsu et al., "Experimental
Medicine (extra number): Basic Technologies in Immunological
Researches", p. 255-, published by Yodosha, 1995].
[0185] 4. The same procedures in steps 1-3 above are repeated for
the mouse antibody light-chain .kappa. gene to finally obtain
drug-sensitive clones which are completely deficient in antibody
heavy-chain and light-chain .kappa..
[0186] 5. Human chromosome #14 (fragment) containing a human
antibody heavy-chain gene and marked with a drug-resistance gene
(e.g., G418 resistance gene) is transferred into the clone from
step 4 above (antibody heavy-chain and light-chain
.kappa.-deficient mouse ES cell) by microcell fusion.
[0187] 6. Human chromosome #2 (fragment) or #22 (fragment) or both
containing a human antibody light-chain gene(s) and marked with a
drug-resistance gene different from the one used in step 5 above
(e.g., puromycin resistance gene) are transferred into the clone
obtained in step 5 above by microcell fusion.
[0188] 7. Chimeric mice are produced from the ES cells obtained in
step 6 above by using embryos obtained from a mouse in a strain
having no ability to produce its own antibody (e.g., RAG-2 knockout
mouse, Shinkai et al., Cell, 68:855-, 1992; membrane-type .mu.
chain knockout mouse, Kitamura et al., Nature, 350:423-, 1991) as
host embryos.
[0189] 8. Most of the functional B lymphocytes in the resultant
chimeric mice are derived from the ES cells [Seiji Takatsu et al.,
"Experimental Medicine (extra number): Basic Technologies in
Immunological Researches", p. 234-, published by Yodosha, 1995].
Since those B lymphocytes are deficient in mouse heavy-chain and
light-chain .kappa., they produce human antibodies alone mainly as
a result of the expression of the functional human antibody genes
on the transferred chromosomes.
[0190] Specific Procedures for Method B
[0191] 1. Chimeric mice retaining a human chromosome or a fragment
thereof containing human antibody heavy-chain, light-chain .kappa.
or light-chain .lambda. are used to produce a progeny which stably
retains the human chromosome or fragment thereof and which can
transmit it to the next generation.
[0192] 2. A mouse in a strain which is homozygous regarding the
deficiency in mouse antibody heavy-chain and light-chain K and
which retains human chromosomes containing human antibody
heavy-chain (#14)+light-chain .kappa. (#2), heavy-chain
(#14)+light-chain .lambda. (#22) or heavy-chain (#14)+light-chain
.kappa. (#2)+light-chain .lambda. (#22) is obtained by mating the
mouse in a strain expressing human antibody heavy-chain or
light-chain from step 1 above or a mouse in a strain expressing
both human antibody heavy and light-chains obtained by mating the
mice from step 1, with a mouse in a strain deficient in its own
antibody genes (e.g., the membrane-type .mu. chain knockout mouse
mentioned above; light-chain .kappa. knockout mouse, Chen et al.,
EMBO J., 3:821-, 1993). Since mice in the resultant strain are
deficient in mouse antibody heavy-chain and light-chain K genes,
they produce human antibodies alone mainly as a result of the
expression of the functional human antibody genes on the
transferred chromosomes.
[0193] Both Method A and Method B may be used not only to yield
human antibodies but also to yield products of any genes located on
a foreign chromosome efficiently.
[0194] The present invention will now be explained in greater
detail with reference to the following examples, which do not limit
the scope of the present invention.
EXAMPLE 1
[0195] Production of Chromosome Donor Cell Retaining Human
Chromosome (Fragment) Labeled with G418 Resistance
[0196] Plasmid pSTneoB containing a G418 resistance gene (Katoh et
al., Cell Struct. Funct., 12:575, 1987; Japanese Collection of
Research Biologicals (JCRB), Deposit Number: VE 039) was linearized
with restriction enzyme SailI (TAKARA SHUZO CO., LTD.) and
introduced into human normal fibroblast cell HFL-1 (obtained from
RIKEN Cell Bank, RCB0251). The HFL-1 cells were treated with
trypsin and suspended in Dulbecco's phosphate-buffered saline (PBS)
at a concentration of 5.times.10.sup.6 cells/ml, followed by
electroporation using a Gene Pulser (Bio-Rad Laboratories, Inc.) in
the presence of 10 .mu.g of DNA (Ishida et al., "Cell Technology
Experiment Procedure Manual", published by Kodansha, 1992). A
voltage of 1000 V was applied at a capacitance of 25 .mu.F with an
Electroporation Cell of 4 mm in length (165-2088, Bio-Rad
Laboratories, Inc.) at room temperature. The electroporated cells
were inoculated into an Eagle's F12 medium (hereinafter referred to
as "F12") supplemented with 15% fetal bovine serum (FBS) in 3-6
tissue culture plastic plates (Corning) of 100 mm.phi.. After one
day, the medium was replaced with a F12 supplemented with 15% FBS
and containing 200 .mu.g/ml of G418 (GENENTICIN, Sigma). The
colonies formed after 2-3 weeks were collected in 52 groups each
consisting of about 100 colonies. The colonies of each group were
inoculated again into a plate of 100 mm.phi. and cultured.
[0197] Mouse A9 cells (Oshimura, Environ. Health Perspect., 93:57,
1991; JCRB 0211) were cultured in Dulbecco's modified Eagle's
medium (hereinafter referred to as "DMEM") supplemented with 10%
FBS in plates of 100 mm.phi.. The G418 resistant HFL-1 cells of 52
groups were cultured in F12 supplemented with 15% FBS and 200
.mu.g/ml of G418 in plates of 100 mm.phi.. The mouse A9 cells and
HFL-1 cells were treated with trypsin and one fourth to one half of
both cells were mixed. The mixed cells were inoculated into a plate
of 100 mm.phi. and cultured in a mixture of equal amounts of DMEM
containing 10% FBS and F12 containing 15% FBS for a period ranging
from a half day to one day. Cell fusion was carried out in
accordance with the method described in Shimizu et al., "Cell
Technology Handbook", published by Yodosha, p.127-, 1992. The cell
surface was washed twice with DMEM and then treated sequentially
with 2 ml of a PEG (1:1.4) solution for 1 minute and with 2 ml of
PEG (1:3) for 1 minute. After the PEG solution was sucked up, and
the cells were washed three times with a serum-free DMEM, followed
by cultivation in DMEM supplemented with 10% FBS for 1 day. The
cells were dispersed by treatment with trypsin and suspended in a
double selective medium (10% FBS supplemented DMEM) containing
ouabain (1.times.10.sup.-5 M, Sigma) and G418 (800 .mu.g/ml),
followed by inoculation in 3 plates of 100 mm.phi.. After about 3
weeks cultivation, the colonies formed were treated with trypsin to
disperse the cells, which were cultured in a selective medium (10%
FBS supplemented DMEM) containing G418 (800 .mu.g/ml).
[0198] The cells were dispersed by treatment with trypsin and two
groups of the cells were collected, followed by cultivation in 6
centrifuge flasks (Coaster, 3025) of 25 cm.sup.2 until the cell
density reached 70-80% confluence. The medium was replaced with a
medium (20% FBS supplemented DMEM) containing Colcemid (0.05
.mu.g/ml, Demecolcine, Wako Pure Chemicals Co., Ltd) and the cells
were cultured for 2 days to form microcells. After the culture
medium was removed, a cytochalasin B (10 .mu.g/ml, Sigma) solution
preliminarily warmed at 37.degree. C. was filled in the 25 cm
.sup.2 centrifuge flask, which were inserted into an acryl
centrifuge container, followed by centrifugation at 34.degree. C.
at 8,000 rpm for 1 hour. The microcells were suspended in a
serum-free medium and purified by passage through a filter. To the
mouse A9 cells cultured to 80% confluence in the flask of 25
cm.sup.2, the purified micorcells were added and the two kinds of
cells were fused with a PEG solution. The fused cells were cultured
in a G418 containing selective medium and colonies formed were
isolated. Human chromosomes #2, 4, 14 and 22 retained in the
respective clones were identified by the methods described in
(1)-(3) below. All other experimental conditions such as operating
procedures and reagents-were in accordance with Shimizu et al.,
"Cell Technology Handbook", published by Yodosha, p127-.
[0199] (1) PCR Analysis
[0200] The isolated cells were cultured and genomic DNA was
extracted from the cells with a Puregene DNA Isolation kit (Gentra
System Co.). PCR was performed using the genomic DNA as a template
with human chromosome specific primers to select the clones
retaining human chromosome #2, 4, 14 or 22. The PCR amplification
was conducted with about 0.1.mu.g of the genomic DNA as a template,
using a thermal cycler (GeneAmp 9600, Perkin-Elmer Corp.) in
accordance with the method described in Innis et al., "PCR
Experiment Manual", published by HBJ Publication Office, 1991. Taq
polymerase was purchased from Perkin-Elmer Corp. and the reaction
was performed in a cycle of 94.degree. C., 5 minutes and 35 cycles
of denaturing at 94.degree. C., 15 seconds, annealing at
54-57.degree. C., 15 seconds (variable with the primers) and
extension at 72.degree. C., 20 seconds. The gene on each chromosome
(O'Brien, Genetic Maps, 6th edition, Book 5, Cold Spring Harbor
Laboratory Press, 1993) and polymorphic markers (Polymorphic STS
Primer Pair, BIOS Laboratories, Inc.; Weissenbach et al., Nature
359:794, 1992; Walter et al., Nature Genetics, 7:22, 1994) were
used as primers. The primers for the genes were prepared on the
basis of nucleotide sequences obtained from data bases such as
GenBank, EMBL and the like. The names of the polymorphic primers
and the sequences of the primers for the genes will be shown for
the respective chromosomes in the following examples (#2, Example
1; #4, Example 6, #14, Example 9; #22, Example 2). The following
genetic markers and polymorphic makers (Polymorphic STS Primer
Pairs: D2S207, D2S177, D2S156 and D2S159, BIOS Laboratories, Inc.)
were used to identify chromosome #2.
[0201] C.kappa. (immunoglobulin kappa constant):
5'-TGGAAGGTGGATAACGCCCT (SEQ ID NO: 1), 5'-TCATTCTCCTCCAACATTAGCA
(SEQ ID NO: 2)
[0202] FABP1 (fatty acid binding protein-1 liver):
5'-GCAATCGGTCTGCCGGAAGA (SEQ ID NO: 3), 5'-TTGGATCACTTTGGACCCAG
(SEQ ID NO: 4)
[0203] Vk3-2 (immunoglobulin kappa variable):
5'-CTCTCCTGCAGGGCCAGTCA (SEQ ID NO: 5), 5'-TGCTGATGGTGAGAGTGAACTC
(SEQ ID NO: 6)
[0204] Vk1-2 (immunoglobulin kappa variable):
5'-AGTCAGGGCATTAGCAGTGC (SEQ ID NO: 7), 5'-GCTGCTGATGGTGAGAGTGA
(SEQ ID NO: 8)
[0205] (2) Fluorescence in situ Hybridization (FISH)
[0206] FISH analysis was conducted with probes specific to human
chromosomes #2, 4, 14 and 22 (CHROMOSOME PAINTING SYSTEM, Cambio
Ltd.) in accordance with the method described in Matsubara et al.,
"FISH Experiment Protocol", published by Shujunsha, 1994.
[0207] For example, at least one clone retaining chromosome #2 was
obtained in 10 groups out of 26 groups (745 clones). Among them,
only 5 clones were positive to all the used primers specific to
chromosome #2. FISH analysis was conducted with these clones. FISH
analysis was conducted with probes specific to human chromosomes #2
(CHROMOSOME PAINTING SYSTEM, Cambio Ltd.) in accordance with the
method described in Matsubara et al., "FISH Experiment Protocol",
published by Shujunsha, 1994. In the cells positive to all the
primers, an intact form of human chromosome #2 was observed. In
some of the clones positive to part of the primers, an independent
chromosome smaller than human chromosome #2 was observed or a cell
having a chromosome in a form fusing with chromosomes other than
human chromosome #2 was observed (FIG. 1). In FIG. 1, the names of
the clones are shown in the horizontal line and the primers used in
the PCR are shown in the left longitudinal line. .circle-solid.
shows positive clones and X shows negative clones. The forms of
human chromosome #2 observed by FISH are shown in the bottom line.
No description means no performance of experiment.
[0208] A9 cells retaining human chromosomes #4, 14 and 22 were
obtained by the same procedure.
EXAMPLE 2
[0209] Transfer of Human Chromosome #22 into Mouse ES cells by
Microcell Fusion
[0210] The mouse A9 cell clones retaining human chromosome #22
(hereinafter referred to as "A9/#22") from Example 1 were used as
chromosome donor cells. Mouse ES cell line E14 (obtained from
Martin L. Hooper; Hooper et al., Nature, 326:292, 1987) was used as
a chromosome recipient cell. E14 cells were cultured in accordance
with the method described in Aizawa Shinichi, "Biomanual Series 8,
Gene Targeting", published by Yodosha, 1995 and G418 resistant STO
cell line (obtained from Prof. Kondo Hisato, Osaka University)
treated with mitomycin C (Sigma) was used as a feeder cell. In the
first step, microcells were prepared from about 10.sup.8 cells of
A9/#22 in accordance with the method reported by Shimizu et al.
"Cell Technology Handbook", published by Yodosha, 1992. The total
amount of the resulting microcells were suspended in 5 ml of DMEM.
About 10.sup.7 cells of E14 were dispersed with trypsin and washed
three times with DMEM and suspended in 5 ml of DMEM. The cells were
then mixed with the microcells and the mixture was centrifuged at
1,250 rpm for 10 minutes to remove the supernatant. The precipitate
was dispersed by tapping and 0.5 ml of a PEG solution (1:1.4) [5 g
of PEG 1000 (Wako Pure Chemicals Co., Ltd.) and 1 ml of DMSO
(Sigma) as dissolved in 6 ml of DMEM) was added. The mixture was
left to stand at room temperature for 1 minute and 30 seconds and
10 ml of DMEM was added slowly. Immediately thereafter, the
resulting mixture was centrifuged at 1,250 rpm for 10 minutes to
remove the supernatant. The precipitate was suspended in 30 ml of a
medium for ES cells and inoculated into 3 tissue culture plastic
plates (Corning) of 100 mm in diameter into which feeder cells were
inoculated. After 24 hours, the medium was replaced with a medium
supplemented with 300 .mu.g/ml of G418 (GENETICIN, Sigma) and
medium replacements were thereafter conducted daily. Drug resistant
colonies appeared in 1 week to 10 days. The frequency of appearance
was 0-5 per 10.sup.7 of E14 cells. The colonies were picked up and
grown. The cells were suspended in a storage medium (a medium for
ES cells+10% DMSO (Sigma)) at a concentration of 5.times.10.sup.6
cells per ml and stored frozen at -80.degree. C. At the same time,
genomic DNA was prepared from 10.sup.6-10.sup.7 cells of each drug
resistant clone with a Puregene DNA Isolation Kit (Gentra System
Co.).
[0211] Human chromosome #22 was fragmented by irradiating the
microcells with .gamma. rays (Koi et al., Science, 260:361, 1993).
The microcells obtained from about 10.sup.8 cells of A9/#22 were
suspended in 5 ml of DMEM and irradiated with .gamma. rays of 60 Gy
on ice with a Gammacell 40 (Canadian Atomic Energy Public
Corporation) at 1.2 Gy/min for 50 minutes. The fusion of .gamma.
ray-irradiated microcells and the selection of drug resistant
clones were conducted by the same procedure as in the case of the
unirradiated microcells. As a result, the frequency of the
appearance of the drug resistant clones was 1-7 per 10.sup.7 of E14
cells. The drug resistant clones were stored frozen and DNA was
prepared from the clones by the same procedure as in the case of
the unirradiated microcells.
[0212] The retention of the transferred chromosomes in the
unirradiated microcell-transferred drug resistant clones E14/#22-9
and E14/#22-10, and in the .gamma. ray-irradiated
microcell-transferred drug resistant clones E14/#22-14 and
E14/#22-25 was confirmed by the methods described in (1)-(3)
below.
[0213] (1) PCR Analysis (FIG. 2)
[0214] The presence of the gene on human chromosome #22 (Genetic
Maps, supra) and polymorphic markers (Polymorphic STS Primer Pairs:
D22S315, D22S275, D22S278, D22S272 and D22S274, BIOS Laboratories,
Inc.; Nature 359:794, 1992) was detected by a PCR method using the
genomic DNA of the drug resistant clone as a template. The
sequences of oligonucleotide primers for the genes prepared on the
basis of nucleotide sequences obtained from data bases such as
GenBank, EMBL and the like are described below.
[0215] PVALB (parvalbumin): 5'-TGGTGGCTGAAAGCTAAGAA (SEQ ID NO: 9),
5'-CCAGAAGAATGGTGTCATTA (SEQ ID NO: 10)
[0216] MB (myoglobin) 5'-TCCAGGTTCTGCAGAGCAAG (SEQ ID NO: 11),
5'-TGTAGTTGGAGGCCATGTCC (SEQ ID NO:
[0217] DIA1 (cytochrome b-5 reductase): 5'-CCCCACCCATGATCCAGTAC
(SEQ ID NO: 13), 5'-GCCCTCAGAAGACGAAGCAG (SEQ ID NO: 14)
[0218] Ig.lambda. (immunoglobulin lambda):
5'-GAGAGTTGCAGAAGGGGTGACT (SEQ ID NO: 15),
5'-GGAGACCACCAAACCCTCCAAA (SEQ ID NO: 16)
[0219] ARSA (arylsulfatase A): 5'-GGCTATGGGGACCTGGGCTG (SEQ ID NO:
17), 5'-CAGAGACACAGGCACGTAGAAG (SEQ ID NO: 18)
[0220] PCR amplification (Innis et al., supra) was conducted by
using about 0.1 .mu.g of the genomic DNA as a template with the
above 10 kinds of the primers. As a result, amplification products
having expected lengths were detected with all the primers in the
case of the two unirradiated clones and with part of the primers in
the case of the .gamma. ray-irradiated two clones. The results are
shown in FIG. 2. In FIG. 2, a schematic chromosome map based on the
G bands of human chrosome #22 and the location of some markers on
bands are shown at the left side (O'Brien, GENETIC MAPS, 6th
edition, BOOK 5, etc.). The arrangement of the genetic and
polymorphic markers shows approximate positional relationships on
the basis of the presently available information (Science, HUMAN
GENETIC MAP, 1994; Nature Genetics, 7:22, 1994; Nature 359:794,
1992, etc.) and the order is not necessarily correct. With respect
to four kinds of the G418 resistant E14 cell clones, the markers
for which the expected amplification products were detected by PCR
are shown by .box-solid. and the markers for which the expected
amplification products were not detected are shown by .quadrature..
The results of the observation by FISH analysis are shown at the
bottom side. A9/#22 is a chromosome donor cell.
[0221] (2) Southern Blot Analysis
[0222] Southern blot analysis of about 2.mu.g of the genomic DNA
digested with restriction enzyme BglII (TAKARA SHUZO CO., LTD.) was
conducted by using human specific repeated sequence L1
(10.sup.4-10.sup.5 copies were present per haploid genome, obtained
from RIKEN DNA Bank; Nucleic acids research, 13;7813, 1985; pUK19A
derived EcoRI-BamHI fragment of 1.4 kb) as a probe in accordance
with the method described in Ausubel et al., Current Protocols in
Molecular Biology, John Wiley & Sons, Inc., 1994. As a result,
a large number of bands hybridized with the human L1 sequence were
detected in DNA of each drug resistant clone. With respect to the
unirradiated 2 clones, their patterns and the quantitative ratio of
human chromosomal DNA to mouse genomic DNA which could be presumed
from the density of the respective bands were the same as those of
A9/#22. The total signal intensity of the bands of the .gamma.-ray
irradiated clones correlated with the degree of the deletion
confirmed by the PCR analysis, as compared with that of A9/#22.
[0223] (3) Fluorescence in situ Hybridization (FISH)
[0224] FISH analysis was conducted with probes specific to human
chromosomes #22 (CHROMOSOME PAINTING SYSTEM, Cambio Ltd.) in
accordance with the method described in Matsubara et al., "FISH
Experiment Protocol", published by Shujunsha, 1994. As a result, in
almost all of the observed metaphase spreads, human chromosome #22
was detected in the form of translocation to the mouse chromosome
with respect to E14/#22-9 and in the form of an independent
chromosome with respect to the three other clones.
[0225] The results of the above experiments demonstrate that the
obtained G418 resistant clones E14/#22-9 and E14/#22-10 retained
all or most part of human chromosome #22 whereas the clones
E14/#22-14 and E14/#22-25 retained partial fragments of human
chromosome #22.
EXAMPLE 3
[0226] Production of Chimeric Mice from the ES Cells Retaining
Human Chromosome #22
[0227] General procedures for obtaining mouse embryos, cultivation,
injection of the ES cells into the embryos, transplantation to the
uteri of foster mothers were carried out in accordance with the
method described in Aizawa Shinichi, "Biomanual Series 8, Gene
Targeting", published by Yodosha, 1995. The cells in a frozen stock
of the G418 resistant ES clone E14/#22-9 which was confirmed to
retain human chromosome #22 were thawed, started to culture and
injected into blastcyst-stage embryos obtained by mating a
C57BL/6.times.C3H F1 female mouse (CREA JAPAN, INC.) with a C3H
male mouse (CREA JAPAN, INC.); the injection rate was 10-15 cells
per embryo. Two and half days after a foster mother [ICR or
MCH(ICR)] mouse (CREA JAPAN, INC) was subjected to a pseudopregnant
treatment, about ten of the ES cell-injected embryos were
transplanted to each side of the uterus of the foster mother. The
results are shown in Table 1.
1TABLE 1 Production of chimeric mice from the ES cells retaining
human chromosome #22 (fragments) Number of ES ES cell G418
cell-injected Number of Number of Contribution clone/human
resistant blastocyst offspring chimeric to coat color chromosome
clone No. stage embryos mice mice <-10% 10-30% 30%< E14/#22 9
166 29 16 7 3 6
[0228] As a result of the transplantation of a total 166 of
injected embryos, 29 offspring mice were born. Chimerism in the
offsprings can be determined by the extent of E14 cell-derived pale
gray coat color in the host embryo-derived agouti coat color (dark
brown). Out of the 29 offsprings, 16 mice were recognized to have
partial pale gray coat color, indicating the contribution of the
E14 cells. The maximum contribution was about 40% in K22-22.
[0229] These results show that the mouse ES cell clone E14/#22-9
retaining human chromosome #22 maintains the ability to produce
chimera, that is, the ability to differentiate into normal tissues
of mouse.
EXAMPLE 4
[0230] Confirmation of Retention of Human Chromosomal DNA in
Various Tissues of the Chimeric Mice Derived from the ES Cells
Retaining Human Chromosome #22
[0231] In addition to the determination of coat color in Example 3,
the retention of the transferred chromosome was confirmed by PCR
analysis using a template genomic DNA prepared from the tail of the
chimeric mouse. The tail was obtained from the chimeric mouse at
least 3 weeks old in accordance with the method described in Motoya
Katsuki, "Development Technology Experiment Manual", published by
Kodansha Scientific, 1987. Genomic DNA was extracted from the tail
with a Puregene DNA Isolation Kit. Out of the polymorphic primers
used in Example 2, PVALB and D22S278 were used, with the extracted
genomic DNA as a template, to confirm the amplification products.
The analysis was conducted with 10 of the mice in which the
contribution to coat color was observed. As a result, the products
of amplification with at least either of the primers were detected
in all the mice.
[0232] Southern blot analysis was conducted in the same manner as
in Example 2 by using human L1 sequence as a probe with 2 .mu.g of
the genomic DNA derived from the tails of the 6 chimeric mice and
one non-chimeric mouse. As a result, the presence of a large number
of human L1 sequence was observed in all the chimeric mice and
their patterns were similar to those of E14/#22-9. The quantitative
ratio to mouse genome was about 10% at maximum (FIG. 3). In FIG. 3,
2 .mu.g of genomic DNA digested with BglII was used in each lane.
Human L1 sequence labeled with .sup.3 2P was used as a probe and
signals were detected with Image Analyzer BAS2000 (Fuji Photo Film
Co., Ltd.). The lanes represent the genomic DNA derived from the
tails of the chimeric mice (K22-6, 7, 8, 9, 10, 11 and 12; 9 is the
non-chimeric mouse) and control DNA (C which is a mixture of
E14/#22-9 genomic DNA and E14 genomic DNA at a weight ratio of 1:9)
as counted from the right. The DNA molecular weights are shown at
the left side and chimerism in the chimeric mice at the right side
(-: 0%, +: <10%, and ++: 10-30%).
[0233] With respect to the chimeric mouse (K22-7) having about 5%
contribution to coat color, genomic DNA was obtained from the
brain, liver, muscle, heart, spleen, thymus, ovary and kidney with
an ISOGEN (Nippon Gene Co.). For each tissue, PCR analysis was
conducted with MB and D1A1 selected from the primers for the genes
used in Example 2. As a result, both primers gave expected
amplification products in all the tissues. The results of PCR
analysis using DlA1 primer are shown in FIG. 4. The PCR products
were electrophoresed on a 2% agarose gel and stained with ethidium
bromide for detection. The lanes in FIG. 4 represent the following
from the left: B, brain; L, liver; SM, skeletal muscle; H, heart;
Sp, Spleen; Th, thymus; Ov, ovary; K, kidney; nc, non-chimeric
mouse tail-derived DNA (negative control); pc, human fibroblast
cell (HFL-1) DNA (positive control).
[0234] These results show that E14/#22-9 contributed to various
normal tissues in the mouse and that it retained human chromosome
#22.
EXAMPLE 5
[0235] Expression of the Human Genes in the Chimeric Mouse Derived
from the ES Cell Retaining Human Chromosome #22
[0236] The tail of the mouse (K22-7) having about 5% contribution
to coat color was frozen with liquid nitrogen and then disrupted
for use as a sample for confirming the expression of the human
genes. The sample was a mixture of tissues such as skin, bones,
muscles, blood and the like. Total RNA was extracted from the
sample with an ISOGEN (Nippon Gene Co.) and used in an RT-PCR
method to detect mRNAs of human myoglobin (MB) and human cytochrome
b5 reductase (DlA1). The RT-PCR was performed in accordance with
the method described in Innis et al., "PCR Experiment Manual",
published by HBJ Publication Office, 1991. Randam hexamer
oligonucleotides (final concentration: 100 pmol, TAKARA SHUZO CO.,
LTD.) were used as primers for reverse transcription and Super
Script (BRL Co.) as reverse transcriptase. The following primers
were used for amplification using cDNA as a template.
2 MB: 5'-TTAAGGGTCACCCAGAGACT, (SEQ ID NO:19)
5'-TGTAGTTGGAGGCCATGTCC (SEQ ID NO:20) DIA1:
5'-CAAAAAGTCCAACCCTATCA, (SEQ ID NO:21) 5'-GCCCTCAGAAGACGAAGCAG
(SEQ ID NO:22)
[0237] As a result, amplification products specific to mRNAs of
both genes were detected (FIG. 5). The RT-PCR products were
electrophoresed on a 2% agarose gel and stained with ethidium
bromide for detection. In FIG. 5, M is a marker (HindIII digested
.lambda. DNA+HaeIII digested .phi. X174DNA, TAKARA SHUZO CO.,
LTD.); MB, human myoglobin; DlA1, human cytochrome b5 reductase;
and WT, a wild-type C3H mouse.
[0238] With respect to the same individual (K22-7), total RNA was
extracted from the brain, heart, thymus, liver, spleen, kidney,
ovary and skeletal muscle with an ISOGEN and RT-PCR was performed
on each organ with the above two primers. As a result, expected
products of amplification with DlA1 were observed in all the organs
and those with MB were observed only in the heart and skeletal
muscle (FIG. 6). Myoglobin is known to be expressed specifically in
muscle cells (Bassel-Duby et al., MCB, 12:5024, 1992). Hence, the
above results show that the gene on the transferred human
chromosome can be subjected to the normal tissue-specific
regulation in the mouse. The PCR products were electrophoresed on a
2% agarose gel and stained with ethidium bromide for detection. In
FIG. 6, the lanes represent the following from the left: B, brain;
H, heart; Th, thymus; L, liver; Sp, spleen; K, kidney; Ov, ovary;
SM, skeletal muscle; and M, marker (supra). The lower band observed
in the results of MB are believed to represent non-specific
products.
[0239] These results show that the transferred human chromosome #22
can function in normal tissues of the chimeric mice.
EXAMPLE 6
[0240] Transfer of Human Chromosome #4 or Fragments Thereof into ES
Cells
[0241] The mouse A9 cell clone retaining human chromosome #4
(hereinafter referred to as "A9/#4") from Example 1 was used as a
chromosome donor cell. Mouse ES cell line E14 (see Example 2) was
used as a chromosome recipient cell. The microcell fusion and the
selection of G418 resistant clones were conducted by the same
procedures as in Example 2. The frequency of the appearance of the
drug resistant clones was 1-2 per 10.sup.7 of E14 cells. The drug
resistant clones were stored frozen and genomic DNA were prepared
by the same procedures as in Example 2. The retention of the
transferred human chromosome #4 or fragments thereof in the drug
resistant clones E14/#4-4, E14/#4-7 and E14/#4-11 was confirmed by
the methods described in (1)-(3) below.
[0242] (1) PCR Analysis (FIG. 7)
[0243] The presence of the gene on human chromosome #4 (O'Brien,
Genetic Maps, 6th edition, Book 5, Cold Spring Harbor Laboratory
Press, 1993) and polymorphic markers (Polymorphic STS Primer Pairs:
D4S395, D4S412, D4S422, D4S413, D4S418, D4S426 and F11, BIOS
Laboratories, Inc.; Nature 359:794, 1992) was detected by a PCR
method. The sequences of oligonucleotide primers for the genes
prepared on the basis of nucleotide sequences obtained from data
bases such as GenBank, EMBL and the like will be described
below.
3 HD (huntington disease): 5'-TCGTTCCTGTCGAGGATGAA, (SEQ ID NO:23)
5'-TCACTCCGAAGCTGCCTTTC (SEQ ID NO:24) IL-2 (interleukin-2):
5'ATGTACAGGATGCAACTCCTG, (SEQ ID NO:25) 5'-TCATCTGTAAATCCAGCAGT
(SEQ ID NO:26) KIT (c-kit): 5'GATCCCATCGCAGCTACCGC, (SEQ ID NO:27)
5'-TTCGCCGAGTAGTCGCACGG (SEQ ID NO:28) FABP2 (fatty acid binding
protein 2, intestinal), 5'-GATGAACTAGTCCAGGTGAGTT, (SEQ ID NO:29)
5'CCTTTTGGCTTCTACTCCTTCA (SEQ ID NO:30)
[0244] PCR amplification was conducted with the above 11 kinds of
the primers. As a result, the amplification products having
expected lengths were detected with all or part of the primers in
all the three clones. In the E14/#4-4 and E14/#4-7 clones, the
deletion of partial regions was observed. The results are shown in
FIG. 7. In FIG. 7, a schematic chromosome map based on the G bands
of human chromosome #4 and the location of some markers on bands
are shown at the left side (see Example 2). The arrangement of the
genetic and polymorphic markers shows approximate positional
relationships on the basis of the presently available information
(see Example 2) and the order is not necessarily correct. With
respect to the three kinds of the G418 resistant E14 cell clones,
the markers for which the expected amplification products were
detected are shown by .box-solid. and the markers for which the
expected amplification products were not detected are shown by
.quadrature.. The results of the observation by FISH analysis are
shown at the lower side. A9/#4 is a chromosome donor cell.
[0245] (2) Southern Blot Analysis (FIG. 8)
[0246] Southern blot analysis was conducted by the same procedure
as in Example 2 using human L1 sequence as a probe with genomic
DNAs obtained from E14/#4-4 and E14/#4-7. As a result, a large
number of bands hybridized with the human L1 sequence were detected
in DNAs of both drug resistant clones. The total signal intensity
correlated with the degree of the deletion confirmed by the PCR
analysis, as compared with that of A9/#4. In FIG. 8, 2 .mu.g of
genomic DNA digested with BglII was used in each lane. Human L1
sequence labeled with .sup.3 2P was used as a probe and the signals
were detected with an Image Analyzer (BAS 2000, Fuji Photo Film
Co., Ltd.). In FIG. 8, the lanes represent the following as counted
from the left: 1, A9/#4 (chromosome donor cell); 2, A9/#4+A9 (1:2);
3, A9/#4+A9 (1:9); 4, A9; 5, E14/#4-7; and 6, E14/#4-4. Lanes 2 and
3 represent mixtures of two kinds of DNAs at the ratios shown in
parentheses. The molecular weights of DNAs are shown at the left
side.
[0247] (3) Fluorescence in situ Hybridization (FISH)
[0248] FISH analysis was conducted with probes specific to human
chromosomes #4 (CHROMOSOME PAINTING SYSTEM, Cambio Ltd.) by the
same procedure as in Example 2. As a result, in almost all of the
observed metaphase spreads of the three clones used, human
chromosome #4 or partial fragments thereof were detected in the
form of translocation to the mouse chromosome with respect to
E14/#4-4 and in the form of an independent chromosome with respect
to the two other clones. The relative sizes of the observed human
chromosome were consistent with those presumed from the results of
the PCR analysis.
[0249] The results of the above experiments demonstrate that the
obtained G418 resistant clones retained the whole human chromosome
#4 or partial fragments thereof.
EXAMPLE 7
[0250] Production of Chimeric Mice from the ES Cells Retaining
Human Chromosome #4 Fragments
[0251] The cells in frozen stocks of the G418 resistant ES cell
clones E14/#4-4 and E14/#4-7 which were confirmed to retain partial
fragments of human chromosome #4 were thawed, started to culture,
and injected into blastcyst stage embryos obtained by the same
method as in Example 3; the injection rate was 10-15 cells per
embryo. Two and half days after a foster mother [ICR or MCH(ICR)]
mouse (CREA JAPAN, INC.) was subjected to a pseudopregnant
treatment, about ten of the ES cell-injected embryos were
transplanted to each side of the uterus of the foster mother. The
results are shown in Table 2.
4TABLE 2 Production of chimeric mice from the E14 cell clones
retaining human chromosome #4 (fragments) Number of ES ES cell G418
cell-injected Number of Number of Contribution clone/human
resistant blastocyst offspring chimeric to coat color chromosome
clone No. stage embryos mice mice <10% 10-30% 30%< E14/#4 4
160 8 5 5 -- -- 7 80 5 2 1 1 --
[0252] As a result of the transplantation of a total of 240
injected embryos, 13 offspring mice were born. Chimerism in the
offsprings can be determined by the extent of E14 cell-derived pale
gray coat color in the host embryo-derived agouti coat color (dark
brown). Out of the 13 offsprings, 7 mice were recognized to have
partial pale gray coat color, indicating the contribution of the
E14 cells. The maximum contribution was about 15% in one individual
derived from E14/#4-7.
[0253] These results show that the mouse ES cell clones E14/#4-4
and E14/#4-7 which retain fragments of human chromosome #4 maintain
the ability to produce chimera, that is, the ability to
differentiate into normal tissues of mouse.
EXAMPLE 8
[0254] Confirmation of Retention of Human Chromosomal DNA in the
Chimeric Mice Derived from the ES Cells Retaining Partial Fragments
of Human Chromosome #4 and Expression of the G418 Resistance
Gene
[0255] (1) PCR Analysis
[0256] Using the chimeric mice produced in Example 7, genomic DNAs
were prepared from the tails of one individual derived from
E14/#4-7 (K/#4-7-1: about 5% chimerism) and one individual derived
from E14/#4-4 (K/#4-4-41: about 5% chimerism) by the same procedure
as in Example 4. These DNAs were used as templates to conduct PCR
analysis using polymorphic marker F11 for chromosome #4 analysis
(see Example 6) which was detected in E14/#4-7 and E14/#4-4. As a
result, expected amplification products were detected in both
mice.
[0257] (2) Southern Analysis (FIG. 9)
[0258] Southern analysis was conducted in the same manner as in
Example 2 by using human L1 sequence as a probe with 2 .mu.g of the
genomic DNA derived from the tail of one individual derived from
E14/#4-7 (K/#4-7-1: about 5% chimerism). As a result, the presence
of a large number of human L1 sequence was observed and their
patterns were similar to those of E14/#4-7. The quantitative ratio
to mouse genome was about 10% of that of E14/#4-7 at maximum. In
FIG. 9, 2 .mu.g of genomic DNA digested with BglII was used in each
lane. Human L1 sequence labeled with .sup.32P was used as a probe
and signals were detected with Image Analyzer BAS2000 (Fuji Photo
Film Co., Ltd.). The molecular weights of DNAs are shown at the
left side. The lanes represent the following as counted from the
left: 1, K/#4-7-1; 2, blank; and 3, E14/#4-7.
[0259] (3) Test on the Tail-Derived Fibroblast Cells for G418
Resistance
[0260] Fibroblast cells were prepared from the tails of one
individual derived from E14/#4-7 (K/#4-7-1: about 5% chimerism) and
one individual derived from E14/#4-4 (K/#4-4-41: about 5%
chimerism). In the same procedure as in Example 4, the tail of each
mouse was cut at a length of 5-10 mm and washed several times with
PBS/1 mM EDTA, followed by notching of the tail with a knife. The
outer skin layer was removed and the inner tissues were cut into
fine pieces. The fine pieces of tissues were transferred into a
tube containing 5 ml of PBS/1 mM EDTA and left to stand for 30
minutes to 1 hour at room temperature. Subsequently, the
supernatant was removed leaving a 1 ml portion of the PBS/EDTA
behind, and 1 ml of 0.25% trypsin/PBS was added. The tissues were
dispersed thoroughly by tapping or pipetting at room temperature
for 5-10 minutes. After centrifugation at 1,000 rpm for 10 minutes,
the precipitate was suspended in 2 ml of DMEM (10% FCS) and
inoculated into a 35 mm plate. After cultivation for 7-10 days, the
cells were treated with trypsin and about 10.sup.4 cells per plate
were inoculated into two 35 mm plates. G418 was added to the medium
in one plate at a final concentration of 400 .mu.g/ml. The cells
were cultured for 5-7 days and the appearance of viable cells in
each plate were examined. Under these conditions, 100% of the
wild-type ICR mouse-derived fibroblast cells were killed in the
presence of G418. As a result, G418 resistant fibroblast cells was
present in both mice.
[0261] These results show that E14/#4-7 and E14/#4-4 contributed to
various normal tissues in the mouse and that they retained partial
fragments of human chromosome #4.
EXAMPLE 9
[0262] Transfer of Human Chromosome #14 or Fragments thereof into
Mouse ES Cells
[0263] The mouse A9 cell clone retaining human chromosome #14
(hereinafter referred to as "A9/#14") from Example 1 was used as a
chromosome donor cell. Mouse ES cell line TT2 (purchased from
Lifetech Oriental Co., Yagi et al., Analytical Biochem., 214:70,
1993) was used as a chromosome recipient cell. The TT2 cells were
cultured in accordance with the method described in Aizawa
Shinichi, "Biomanual Series 8, Gene Targeting", published by
Yodosha, 1995 and G418 resistant primary culture cells (purchased
from Lifetech Oriental Co.) treated with mitomycin C (Sigma) were
used as feeder cells. The microcell fusion and the selection of
G418 resistant clones were conducted by the same procedures as in
Example 2. The frequency of the appearance of the drug resistant
clones was 3-6 per 10.sup.7 of TT2 cells. The drug resistant clones
were stored frozen and genomic DNA was prepared by the same
procedures as in Example 2.
[0264] Human chromosome #14 was fragmented by irradiating the
microcells with .gamma.-rays (Koi et al., Science, 260:361, 1993).
The microcells obtained from about 10.sup.8 cells of A9/#14 were
suspended in 5 ml of DMEM and irradiated with .gamma.-rays of 30 Gy
on ice with a Gammacell 40 (Canadian Atomic Energy Public
Corporation) at 1.2 Gy/min for 25 minutes. The fusion of .gamma.
ray-irradiated microcells and the selection of drug resistant
clones were conducted by the same procedure as in the case of the
unirradiated micorcells. As a result, the frequency of the
appearance of the drug resistant clones was 3 per 10.sup.7 of TT2
cells. The drug resistant clones were frozen stored and DNA was
prepared by the same procedure as in Example 2.
[0265] The retention of human chromosome #14 or partial fragments
thereof in the unirradiated microcell-transferred G418 resistant
clones 1-4 and 1-5, and in the G418 resistant clones 3-1 and 3-2 (a
total of 4 clones) into which the .gamma.-ray-irradiated microcell
was transferred was confirmed by the methods described in (1) and
(2) below.
[0266] (1) PCR Analysis (FIG. 10)
[0267] The presence of the gene on human chromosome #14 (O'Brien,
Genetic Maps, 6th edition, Book 5, Cold Spring Harbor Laboratory
Press, 1993) and polymorphic markers (Polymorphic STS Primer Pairs:
D14S43, D14S51, D14S62, D14S65, D14S66, D14S67, D14S72, D14S75,
D14S78, D14S81, and PCI, BIOS Laboratories, Inc.; Nature 359:794,
1992; Nature Genetics, 7:22, 1994) was detected by a PCR method
using genomic DNA of the drug resistant clone as a template. The
sequences of oligonucleotide primers for the genes prepared on the
basis of nucleotide sequences obtained from data bases such as
GenBank, EMBL and the like are described below.
5 NP (nucleoside phosphorylase): 5'-ATAGAGGGTACCCACTCTGG, (SEQ ID
NO:31) 5'-AACCAGGTAGGTTGATATGG (SEQ ID NO:32) TCRA (T-cell receptor
alpha): 5'-AAGTTCCTGTGATGTCAAGC, (SEQ ID NO:33)
5'-TCATGAGCAGATTAAACCCG (SEQ ID NO:34) MYH6 (myosin heavy chain
cardiac): 5'-TGTGAAGGAGGACCAGGTGT, (SEQ ID NO:35)
5'-TGTAGGGGTTGACAGTGACA (SEQ ID NO:36) IGA2 (immunoglobulin alpha-2
constant): 5'-CTGAGAGATGCCTCTGGTGC, (SEQ ID NO:37)
5'-GGCGGTTAGTGGGGTCTTCA (SEQ ID NO:38) IGG1 (immunoglobulin gamma-1
constant): 5'-GGTGTCGTGGAACTCAGGCG, (SEQ ID NO:39)
5'-CTGGTGCAGGACGGTGAGGA (SEQ ID NO:40) IGM (immunoglobulin mu
constant): 5'-GCATCCTGACCGTGTCCGAA, (SEQ ID NO: 41)
5'-GGGTCAGTAGCAGGTGCCAG (SEQ ID NO:42) IGVH3 (immunoglobulin heavy
variable-3): 5'-AGTGAGATAAGCAGTGGATG, (SEQ ID NO:43)
5'-GTTGTGCTACTCCCATCACT (SEQ ID NO:44)
[0268] PCR amplification was conducted using the genomic DNAs of
the 4 drug resistant clones as templates with the above 18 kinds of
the primers by the same procedure as in Example 2. As a result,
expected amplification products were detected with all or part of
the primers. In the drug resistant clones 3-1 and 3-2 obtained by
using the .gamma.-ray irradiated microcells, a tendency for the
deletion of partial regions of chromosome #14 was observed. In the
case where the unirradiated microcells were used, deletion was
observed as in the case of the 1-4 clone. The results are shown in
FIG. 10. In FIG. 10, a schematic chromosome map based on the G
bands of human chromosome #14 and the location of some markers on
bands are shown at the left side (see Example 2). The arrangement
of the genetic and polymorphic markers shows approximate positional
relationships on the basis of the presently available information
(see Example 2) and the order is not necessarily correct. With
respect to four kinds of the G418 resistant TT2 cell clones, the
markers for which the expected amplification products were detected
are shown by .box-solid. and the markers for which the expected
amplification products were not detected are shown by .quadrature..
A9/#14 is a chromosome donor cell. The results of Example 11 (1)
are shown at the right side.
[0269] (2) Fluorescence in situ Hybridization (FISH)
[0270] FISH analysis was conducted with probes specific to human
chromosomes #14 (CHROMOSOME PAINTING SYSTEM, Cambio Ltd.) in
accordance with the method described in Matsubara et al., "FISH
Experiment Protocol", published by Shujunsha, 1994. As a result, in
almost all of the observed metaphase spreads of all the 4 clones,
human chromosome #14 or partial fragments thereof were detected in
the form of an independent chromosome. The relative sizes of the
observed human chromosome were consistent with those presumed from
the results of the PCR analysis.
[0271] The results of the above experiments demonstrate that the
obtained G418 resistant clones 1-4, 1-5, 3-1 and 3-2 retained the
whole or partial fragments of human chromosome #14.
EXAMPLE 10
[0272] Production of Chimeric Mice from the ES Cells Retaining
Human Chromosome #14 or Fragments Thereof
[0273] The cells in the frozen stocks of four G418 resistant ES
cell clones (1-4, 3-1, 3-2 and 1-5) that were prepared in Example 9
and which were confirmed to retain human chromosome #14 or
fragments thereof were thawed, started to culture and injected into
8-cell stage embryos obtained by mating [ICR or MCH(ICR)] male and
female mice (CREA JAPAN, INC.); the injection rate was 8-10 cells
per embryo. The embryos were cultured in an ES medium overnight to
develop to blastocysts. Two and half days after a foster mother ICR
mouse (CREA JAPAN, INC.) was subjected to a pseudopregnant
treatment, about ten of the injected embryos were transplanted to
each side of the uterus of the foster mother. The results are shown
in Table 3.
6TABLE 3 Production of chimeric mice from the TT2 cell clones
retaining human chromosome #14 (fragments) Number of ES ES cell
G418 cell-injected Number of Number of Contribution clone/human
resistant 8-cell offspring chimeric to coat color chromosome clone
No. stage embryos mice mice <20% 20-50% 50-80% TT2/#14 1-4 98 20
1 -- -- 1 1-5 110 14 2 1 -- 1 3-1 103 11 2 1 1 -- 3-2 183 19 3 -- 2
1
[0274] As a result of the transplantation of a total of 494
injected embryos, 64 offspring mice were born. Chimerism in the
offsprings can be determined by the extent of TT2 cell-derived
agouti coat color (dark brown) in the host embryo-derived albino
coat color. Out of the 64 produced offsprings, 8 mice were
recognized to have partial agouti coat color, indicating the
contribution of the ES cells. The maximum contribution was about
80% in one individual derived from 1-4.
[0275] These results show that the G418 resistant ES cell clones
(1-4, 1-5, 3-1 and 3-2) retaining human chromosome #14 or fragments
thereof maintain the ability to produce chimera, that is, the
ability to differentiate into normal tissues of mouse.
EXAMPLE 11
[0276] Confirmation of Retention of Human Chromosome #14 Fragment
DNA in the Chimeric Mice Derived from the ES Cells Retaining Human
Chromosome #14 Fragments
[0277] The retention of human chromosome #14 partial fragments in
the chimeric mice obtained in Example 10 was confirmed by the
methods described in (1)-(3) below.
[0278] (1) PCR Analysis using DNAs Derived from Various Tissues
[0279] Genomic DNA was extracted from the tail of one individual
derived from 3-1 (K3-1-1: about 25% chimerism) by the same
procedure as in Example 4. The DNA was used as a template to
conduct PCR analysis using all of the 14 primers for chromosome #14
analysis which were detected in 3-1. As a result, expected
amplification products were detected with all the 14 primers. (FIG.
10)
[0280] With respect to the same individual (K3-1-1), genomic DNA
was obtained from the brain, kidney, spleen, heart, liver and
thymus with a Puregene DNA Isolation Kit. For each tissue, PCR
analysis was conducted with IGM primers (see Example 9). As a
result, expected amplification products were detected in all the
tissues (FIG. 11). The PCR products were electrophoresed on a 2%
agarose gel and stained with ethidium bromide for detection. In
FIG. 11, the lanes represent the following as counted from the
left: B, brain; K, kidney; Sp, Spleen; H, heart; L, liver; Th,
thymus; pc, human fibroblast cell (HFL-1) DNA (positive control);
nc, non-chimeric mouse tail DNA (negative control); and M, marker
(HindIII digested .lambda. DNA+HaeIII digested .phi. X174 DNA,
TAKARA SHUZO CO., LTD.).
[0281] (2) Test on the Tail-Derived Fibroblast Cells for G418
Resistance
[0282] Fibroblast cells were prepared from the tails of two
individuals derived from 3-2 (K3-2-1: about 25% chimerism, and
K3-2-3: about 50% chimerism) and one individual derived from 1-4
(K1-4-1: about 80% chimerism). In the same procedure as in Example
4, the tail of each chimeric mouse of 3-6 weeks was cut at a length
of 5-10 mm and washed several times with PBS/1 mM EDTA, followed by
notching of the tail with a knife. The outer layer was removed and
the inner tissues were cut into fine pieces. The fine pieces of
tissues were transferred into a tube containing 5 ml of PBS/1 mM
EDTA and left to stand for 30 minutes to 1 hour at room
temperature. Subsequently, the supernatant was removed leaving a 1
ml portion of the PBS/EDTA behind, and 1 ml of 0.25% trypsin/PBS
was added. The tissues were dispersed thoroughly by tapping or
pipetting at room temperature for 5-10 minutes. After
centrifugation at 1,000 rpm for 10 minutes, the precipitate was
suspended in 2 ml of DMEM (10% FCS) and inoculated into a 35 mm
plate. After cultivation for 7-10 days, the cells were treated with
trypsin and about 10.sup.4 cells per plate were inoculated into
four 35 mm plates. G418 was added to the medium in two of the
plates at a final concentration of 400 .mu.g/ml. The cells were
cultured for 5-7 days and the viable cells in each plate were
counted. Under these conditions, 100% of the wild-type ICR
mouse-derived fibroblast cells were killed in the presence of G418.
Assuming the same growth rate of the G418 resistant fibroblast in
the non-selective and selective media, the ratio of the viable
cells in the selective medium to those in the non-selective medium
is believed to reflect the contribution in the fibroblast cell
populations of the G418 resistant ES cell-derived fiblablast. As a
result, the presence of G418 resistant fibroblast cells was
observed in all the three individuals as shown in FIG. 12. In FIG.
12, % resistance is an average of 2 pairs of the
selective/non-selective 35 mm plates for each mouse. ICR refers to
the wild-type ICR mice.
[0283] (3) FISH Analysis of the Tail-Derived G418 Resistant
Fibroblast Cells
[0284] FISH analysis of the K3-2-3 and K1-4-1 derived G418
resistant fibroblast cells obtained in (2) was conducted by the
same procedure as in Example 2. Total human DNA extracted from the
HFL-1 cells (Example 1) was labeled with FITC so that is could be
used as a probe (Matsubara et al., "FISH Experimental Protocol",
published by Shujunsha, 1994). As a result, in almost all of the
observed metaphase spreads of the both individuals, partial
fragments of the human chromosome in independent forms were
observed.
[0285] These results show that the TT2 cell clones retaining
fragments of human chromosome #14 contributed to various normal
tissues in the mouse individuals and that they retained partial
fragments of human chromosome #14.
EXAMPLE 12
[0286] Transfer of Partial Fragments of Human Chromosome #2 into ES
Cells
[0287] The mouse A9 cell W23 retaining a human chromosome #2
fragment (hereinafter referred to as "A9/.andgate.2 W23") from
Example 1 was used as a chromosome donor cell. Mouse ES cell line
TT2 (see Example 9) was used as a chromosome recipient cell. The
microcell fusion and the selection of G418 resistant clones were
conducted by the same procedures as in Example 2. The frequency of
the appearance of the drug resistant clones was 1-3 per 10.sup.7 of
TT2 cells. The drug resistant clones were stored frozen and genomic
DNA was prepared by the same procedures as in Example 2. The
retention of partial fragments of human chromosome #2 in drug
resistant clones 5-1, 5-2 and 5-3 was confirmed by the methods
described in (1) and (2) below.
[0288] (1) PCR Analysis
[0289] The presence of C.kappa. and FABP1 that are the genes on
human chromosome #2 (Genetic Maps, supra) and which were detected
in the chromosome donor cell A9/.andgate.2 W23 was detected by a
PCR method.
[0290] As a result of PCR amplification using each primer, expected
amplification products were detected with both primers in all of
the 3 clones.
[0291] (2) Fluorescence in situ Hybridization (FISH)
[0292] FISH analysis was conducted with probes specific to human
chromosome #2 (CHROMOSOME PAINTING SYSTEM, Cambio Ltd.) by the same
method as in Example 2. As a result, in almost all of the observed
metaphase spreads of the 3 clones, partial fragments of human
chromosome #2 in the form of independent chromosomes were detected.
The sizes of the observed human chromosome were the same as those
observed in A9/.andgate.2 W23.
[0293] The results of the above experiments demonstrate that the
obtained G418 resistant clones retained partial fragments of human
chromosome #2.
EXAMPLE 13
[0294] Production of Chimeric Mice from the ES Cells Retaining
Human Chromosome #2
[0295] The cells in a frozen stock of the G418 resistant ES cell
clone 5-1 that was obtained in Example 12 and which was confirmed
to retain human chromosome #2 was thawed, started to culture and
injected into 8-cell stage embryos obtained by mating ICR or
MCH(ICR) male and female mice (CREA JAPAN, INC.); the injection
rate was 10-12 cells per embryo. The embryos were cultured in an ES
medium (Example 9) overnight to develop to blastocysts. Two and
half days after a foster mother ICR mouse (CREA JAPAN, INC.) was
subjected to a pseudopregnant treatment, about ten of the injected
embryos were transplanted to each side of the uterus of the foster
mother. The results are shown in Table 4.
7TABLE 4 Production of chimeric mice from the TT2 cell clone
retaining human chromosome #2 (fragments) Number of ES ES cell G418
cell-injected Number of Number of Contribution clone/human
resistant 8 cell offspring chimeric to coat color chromosome clone
No. stage embryos mice mice <20% 20-50% 50-80% TT2/#2 5-1 264 51
18 7 5 6 (W23)
[0296] As a result of the transplantation of a total of 264
injected embryos, 51 offspring mice were born. Chimerism in the
offsprings can be determined by the extent of TT2 cell-derived
agouti coat color (dark brown) in the host embryo-derived albino
coat color. Out of the 51 produced offsprings, 18 mice were
recognized to have partial agouti coat color, indicating the
contribution of the ES cells. The maximum contribution was about
80%.
[0297] These results show that the G418 resistant ES cell clone
(5-1) retaining a fragment of human chromosome #2 maintains the
ability to produce chimera, that is, the ability to differentiate
into normal tissues of mouse individual.
EXAMPLE 14
[0298] Detection of Human Antibody Heavy Chain in sera of the Human
Chromosome #14 Transferred Chimeric Mice
[0299] The concentrations of human antibody in the sera were
determined by enzyme-linked immunosorbent assay (ELISA). The ELISA
for human antibody was performed in accordance with the method
described in Toyama and Ando, "Monoclonal Antibody Experiment
Manual", published by Kodansha, 1987; Andou and Chiba, "Monoclonal
Antibody Experiment Procedure Manual", published by Kodansha
Scientific, 1991; Ishikawa, "Super High Sensitivity Enzyme Immuno
Assay", published by Gakkai-syuppan center, 1993; Ed Harlow and
David Lane, "Antibodies A Laboratory Manual", published by Cold
Spring Harbor Laboratory, 1988 and A. Doyle and J. B. Griffiths,
"Cell & Tissue Culture: Laboratory Procedures", published by
John Wiley & Sons Ltd., 1996. In some assays, the condition of
reaction were modified, for example, the reaction was performed at
4.degree. C. over night. Antibodies to human-immunogloblin or
antigen were diluted to about 0.5-10 .mu.g/ml (100-5000 fold) and
ELISA plates were coated with these solutions. PBS supplemented
with 5% mouse serum (Sigma, M5905) was used for blocking and
dilution of the samples and labeled antibodies. PBS was used for
20-fold dilution of the chimeric mouse sera. After washed, the
coated plate was blocked over 1 hour. After plate was washed,
sample was added and incubated over a half hour. After washed,
Enzyme labeled anti-human immunogloblin antibodies diluted 100-5000
folds were added to the plates and incubated over 1 hour, the plate
was washed and then substrate was added. In some assays, the same
procedure was applied except that a biotin-labeled antibody was
used. After plate was washed, avidin-enzyme complex was added.
After plate was washed, substrate was added. Absorbances were
measured with a microplate reader (Bio-tek instrument, EL312e). The
chimeric mice (Example 10, K3-1-2, K3-2-2 and K3-2-3) which were
29-35 days old were bled and assayed by ELISA. Anti-human IgM mouse
monoclonal antibody (Sigma, I6385) was diluted with 50 mM
carbonate-bicartonate buffer (pH 9.6) and absorbed to the 96-well
microtiter plates. The serum samples diluted with mouse serum
(Sigma, M5905) supplemented PBS were added to the plates.
Subsequently, peroxidase-labeled anti-human IgM goat antibody
(Tago, 2392) was added and the plates were incubated. After ABTS
substrate (Kirkegaard & Perry Laboratories Inc., 506200) was
added, enzyme activity was determined by absorbance measurement at
405 nm. Purified human IgM antibody (CAPEL, 6001-1590) and IgG
(Sigma, I4506) were used as standards. The standards were diluted
stepwise with mouse serum-supplemented PBS. In the determination of
human IgG concentration, anti-human IgG goat antibody (Sigma,
I3382) was absorbed to the plate and the human IgG was detected
with peroxidase-labeled anti-human IgG goat antibody (Sigma,
A0170). The results are shown in Table 5. Both human IgM and IgG
were detected.
8TABLE 5 Concentrations of Human Antibodies in Chimeric Mouse Sera
(ELISA) Chimeric Mouse IgG (mg/l) IgM (mg/l) K3-1-2 0.37 3.7 K3-2-2
0.33 5.9 K3-2-3 0.51 3.4
[0300] Two milliliters of human serum albumin (HSA, Sigma, A3782)
dissolved in PBS was mixed with adjuvant (MPL+TDM Emulsion, RIBI
Immunochem Research Inc.) to prepare an antigen solution at a
concentration of 0.25 mg/ml. The chimeric mice retaining human
chromosome #14 fragment (Example 10, K3-1-1 and K3-2-1) were
immunized with 0.2 ml of the antigen solution 3 times at days 27,
34 and 41 after birth. The chimeric mouse sera were assayed by
ELISA. The results are shown in FIGS. 13 and 14. The human antibody
concentration in the sera of the HSA-immunized chimeric mice was
increased after the immunization. In the K3-1-1 mouse, 18 .mu.g/mi
of human IgM and 2.6 .mu.g/ml of IgG were detected in the serum at
day 17 after the immunization. In the serum of the control ICR
mouse, the human antibody titer was not significant.
EXAMPLE 15
[0301] Production of Human Antibody Heavy Chain-Producing
Hybridomas from the Human Chromosome #14 Transferred Chimeric
Mouse
[0302] The spleen was removed from the human albumin-immunized
chimeric mouse (K3-1-1, Example 14) at day 44 after birth. The
spleen cell was fused with a myeloma cell to produce a hybridoma.
The hybridoma was produced using a myeloma cell P3.times.63Ag8.653
(DAINIPPON PHARMACEUTICAL CO., LTD., 05-565) by the method
described in Ando and Chiba, "Monoclonal Antibody Experimental
Procedure Manual", published by Kodansha Scientific, 1991. The
hybridomas were inoculated into ten 96-well plates and cultured for
1 week. The culture supernatant was analyzed by ELISA. The ELISA
procedure was conducted by using anti-human IgM mouse monoclonal
antibody (Sigma, I6385) immobilized on ELISA plate in the same
manner as in Example 14 to give 6 positive clones. HSA (antigen)
was dissolved in 50 mM carbonate-bicarbonate buffer (pH 9.6) at a
concentration of 5 .mu.g/ml and the antigen solution was dispensed
in 100 .mu.l portions into all the wells of the ELISA plates. After
the addition of the supernqtant, peroxidase-labeled anti-human
IgA+IgG+IgM goat antibodies (Kierkegaard & Perry Laboratories
Inc., 04-10-17) were used for detection of HSA-specific human
antibody. One positive clone was confirmed in the ten plates. This
clone was one of the 6 human IgM positive clones. The clone (H4B7)
was further cultured and the culture supernatant was diluted,
followed by ELISA analysis using HSA as an antigen with
peroxidase-labeled anti-human IgM goat antibody (Tago, 2392) in the
same manner as described above. As a result, the absorbance
decreased with the increase in the dilution of the culture
solution. Serial twofold dilutions of 2 .mu.g/ml human IgM (CAPEL,
6001-1590) showed low absorbance regardless of dilution ratios.
This suggests that the antibody produced by hybridoma H4B7 had a
specificity to HSA (FIG. 15). In FIG. 15, the dilution of the
culture supernatant samples is plotted on the horizontal axis and
the absorbance at 405 nm is plotted on the vertical axis.
EXAMPLE 16
[0303] Re-Marking of the G418 Resistance-Marked Human Chromosome #2
Fragment with Puromycin Resistance
[0304] The A9 cells retaining the G418 resistance-marked human
chromosome #2 fragment (W23) (see Example 1, FIG. 1) were cultured
in a G418 (800 .mu.g/ml) containing selective medium (10% FBS,
DMEM) in a 100 mm plate. Plasmid pPGKPuro (provided by Dr. Peter W.
Laird (WHITEHEAD INSTITUTE)) containing puromycin resistance gene
was linearized with restriction enzyme SalI (TAKARA SHUZO CO.,
LTD.) before transfection. The cells were treated with trypsin and
suspended in Dulbecco's phosphate buffered saline (PBS) at a
concentration of 5 x 106 cells/ml, followed by electroporation
using a Gene Pulser (Bio-Rad Laboratories, Inc.) in the presence of
10 .mu.g of DNA in the same manner as in Example 1. A voltage of
1000 V was applied at a capacitance of 25 .mu.F with an
Electroporation Cell of 4 mm in length (Example 1) at room
temperature. The electroporated cells were inoculated into media in
3-6 plates of 100 mm.phi.. After one day, the medium was replaced
with a double-selective medium containing 10 .mu.g/ml of puromycin
(Sigma, P-7255) and 800 .mu.g/ml of G418. The colonies formed after
2-3 weeks were collected in groups each consisting of about 200
colonies. The cells of each of the three groups were cultured in
two or three 25 cm.sup.2 flasks to form microcells. The mouse A9
cells were cultured in a 25 cm.sup.2 flask and fused with the
microcells by the same procedure as in Example 1. The fused cells
were transferred into two 100 mm plates and cultured in the
double-selective medium containing G418 and puromycin. One of the
three groups gave two double-drug resistant clones. In these
clones, it was most likely that puromycin resistance marker had
been introduced into human chromosome #2 fragment.
EXAMPLE 17
[0305] Duplication of Transferred Human Chromosome in the Human
Chromosome Transferred ES Cells
[0306] The ES cell clone retaining the G418 resistance marked human
chromosome #14 fragment (E14/#14-36) was cultured in a medium
containing G418 at a high concentration to give ES cell clones in
which the human chromosome was duplicated ("Biomanual Series 8,
Gene Targeting", published by Yodosha, 1995). G418 resistant mouse
primary cells (purchased from Lifetech Oriental) were inoculated
into a 100 mm plate without treating with mitomycin C and used as
feeder cells. The E14/#14-36 cells were inoculated into the 100 mm
plate and after half a day, the medium was replaced with a medium
containing G418 at a concentration of 16 mg/ml. The medium was
replaced every 1-2 days. The G418 concentration was changed to 10
mg/ml one week later and the cultivation was continued. Among the
colonies formed, 15 were picked up and cultured, followed by FISH
analysis of chromosome using human chromosome #14 specific probes
(see Example 9). As a result, human chromosome #14 fragment was
found to have duplicated in the 8 clones.
EXAMPLE 18
[0307] Preparation of Mouse ES Cells Retaining Both Human
Chromosome #2 Partial Fragments and Human Chromosome #14 Partial
Fragments.
[0308] In a microcell transfer experiment using the double-drug
resistant clone PG-1 from Example 16 as a microcell donor cell and
a wild-type A9 cell as a recipient cell, it was confirmed that the
human chromosome #2 partial fragment retained in PG-1 was marked
with a puromycin resistance gene. The preparation of microcells and
the fusion with the A9 cells was carried out by the same methods as
in Example 1. As a result, 10 days after the microcell fusion, a
total of fifty nine G418 resistant colonies appeared. After the
medium for these colonies was changed to one containing 8 .mu.g/ml
puromycin, the colonies were cultured for 3 days to give 45 viable
colonies (76%). In many cases of microcell fusion, only one or few
chromosomes are transferred into a recipient cell. Hence,
cotransfer of both the resistance genes at a high frequency shows
that the G418 resistance-labeled chromosome #2 partial fragment
retained in the PG1 clone was also marked with the puromycin
resistance gene. In addition, for the detection of the respective
marker genes on the human chromosome #2 partial fragment, FISH
analysis was conducted by using pSTneoB (see Example 1) as a probe
in the case of the A9/.andgate.2 W23 clone having only G418
resistance (see Example 16) and by using pPGKPuro (see Example 16)
as a probe in the case of the PG1 clone in accordance with the
method described in Matsubara et al., "FISH Experiment Protocol",
published by Shujunsha, 1994. As a result, in the case of the
A9/.andgate.2 W23 clone, one signal was observed in each of the
sister chromatids of the human chromosome #2 partial fragment
observed in Example 12 (2 signals in total). This indicated the
insertion of pSTneoB into the human chromosome #2 partial fragment
at one site. In the case of the PG1 clone, a total of 4 signals
were observed on a chromosome fragment of the same size as in
A9/.andgate.2 W23. Since pSTneoB and pPGKPuro had identical
sequences in their vector portions, the pSTneoB could be detected
by the pPGKPuro probe. Hence, it is believed that out of the four
signals observed in the PG1 clone, two were from the pSTneoB and
the other two were from the pPGKPuro. These results show that the
human chromosome #2 partial fragment retained in the PG1 was marked
with both the G418 and puromycin resistances.
[0309] The PG1 cell clone was used as a chromosome donor cell to
prepare a mouse ES cell retaining both a human chromosome #2
partial fragment and a human chromosome #14 partial fragment. The
G418 resistant TT2 cell clone 1-4 already retaining the human
chromosome #14 partial fragment (see Example 9) was used as a
chromosome recipient cell. The microcell fusion and the selection
of puromycin resistant cells were carried out by the same methods
as in the selection of the G418 resistant clones in Example 9
except that the concentration of puromycin was 0.75 .mu.g/ml. The
frequency of the appearance of the resulting puromycin resistant
clones was 3-7 per 10.sup.7 of 1-4 cells. The presence of G418
resistance in these puromycin resistant clones was confirmed from
the fact that they were grown in the presence of 300 .mu.g/ml of
G418. The double-drug resistant clones were stored frozen and
genomic DNA was prepared by the same methods as in Example 2. The
retention of the human chromosome #2 partial fragment and human
chromosome #14 partial fragment was confirmed by the method
described in (1) in the case of double-drug resistant clones PG5,
PG15 and PG16 and by the method described in (2) in the case of the
clone PG15.
[0310] (1) PCR Analysis
[0311] Genomic DNAs of the double-drug resistant clones were used
as templates in the PCR amplifications. Among the markers on human
chromosomes #2 and #14 (Genetic Maps, supra), the primers whose
presence in the A9/.andgate.2 W23 clone was confirmed in Example 12
and those whose presence in the TT2/#14 1-4 clone was confirmed in
Example 9 were used. All the primers gave expected amplification
products in all the three clones.
[0312] (2) Fluorescence in situ Hybridization (FISH)
[0313] FISH analysis was conducted by using FITC-labeled human
total DNA as a probe in the same manner as in Example 11. As a
result, in almost all of the metaphase spreads, two (large and
small) human chromosome fragments were detected. The large fragment
had the same size as that of the partial fragment detected by using
the human chromosome #14 specific probes in the case of the TT2/#14
1-4 clone in Example 9 and the small fragment had the same size as
that of the partial fragment detected by using the human chromosome
#2 specific probes in the case of the TT2/.andgate.2 5-1 in Example
12. The results are shown in FIG. 16. In FIG. 16, the less bright
chromosome was derived from the mouse. The two (large and small)
chromosome fragments of high brightness due to FITC fluorescence as
shown by arrows were derived from the human, which are believed to
correspond to the human chromosome #14 and #2 partial
fragments.
[0314] These results show that the obtained double-drug resistant
ES clones retained both the human chromosome #2 partial fragment
and the human chromosome #14 partial fragment.
EXAMPLE 19
[0315] Production of Chimeric Mice from the Mouse ES Cell Clones
Retaining Both Human Chromosome #2 Partial Fragments and Human
Chromosome #14 Partial Fragments
[0316] The cells in frozen stocks of the G418 and puromycin
double-resistant TT2 cell clones PG5, PG15 and PG16 from Example 18
which were confirmed to retain human chromosome #2 partial
fragments and human chromosome #14 partial fragments were thawed,
started to culture and injected into 8-cell stage embryos obtained
by mating ICR or MCH(ICR) male and female mice (CREA JAPAN, INC.);
the injection rate was 10-12 cells per embryo. The embryos were
cultured in a medium for ES cells (see Example 9) overnight to
develop to blastocysts. Two and a half day after a foster mother
ICR mouse was subjected to a pseudopregnant treatment, about ten of
the injected embryos were transplanted to each side of the uterus
of the foster mother. The results are shown in Table 6.
9TABLE 6 Production of chimeric mice from the mouse ES cell clones
retaining both human chromosome #2 partial fragments and human
chromosome #14 partial fragments Number of ES ES cell Double-drug
cell-injected Number of Number of Contribution clone/human
resistant 8-cell stage offspring chimeric to coat color chromosome
clone No. embryos mice mice <10% 10-50% 50%< TT2/#14 + #2 PG5
160 26 8 7 1 -- PG15 168 15 3 1 2 -- PG16 223 32 12 3 6 3
[0317] As a result of the transplantation of a total of 551
injected embryos, 73 offspring mice were born. Chimerism in the
offsprings can be determined by the extent of TT2 cell-derived
agouti coat color (dark brown) in the host embryo-derived albino
coat color. Out of the 73 produced offsprings, 23 mice were
recognized to have a partial agouti coat color, indicating the
contribution of the ES cells.
[0318] These results show that the ES cell clones PG5, PG15 and
PG16 retaining human chromosome #2 partial fragments and human
chromosome #14 partial fragments maintain the ability to produce
chimera, that is, the ability to differentiate into normal tissues
of mouse.
EXAMPLE 20
[0319] Detection of Human Antibody in sera of the Chimeric Mice
Derived from the ES Cells Retaining Both Human Chromosome #2
Partial Fragments and Human Chromosome #14 Partial Fragments
[0320] The two KPG-15 (9 weeks old; derived from the PG-5 clone,
10% chimerism) and KPG-18 (5 weeks old; derived from the PG-5
clone, 10% chimerism) chimeric mice from Example 19 were immunized
with 0.2 ml of a solution of human serum albumin (HSA, Sigma,
A3782) and adjuvant (MPL+TDM Emulsion, RIBI Immunochem Research
Inc.) at a HSA concentration of 0.25 mg/ml. The chimeric mice were
bled just before the immunization and 8 days after that and the
concentrations of human antibody .mu. and .kappa. chains in the
sera were determined by ELISA (see Example 14). Ninety six-well
microtiter plates were coated with anti-human antibody .kappa.
chain goat antibody (VECTOR LABORATORIES INC., AI-3060) diluted
with 50 mM carbonate-bicarbonate buffer (pH 9.6) and then a serum
sample diluted with mouse serum (Sigma, M5905)-containing PBS was
added. Subsequently, biotin-labeled anti-human antibody .kappa.
chain goat antibody (VECTOR LABORATORIES INC., BA-3060) was added
to the plates and incubated. A complex of biotinylated horseradish
peroxidase and avidin DH (VECTOR LABORATORIES, INC., Vectastain ABC
Kit, PK4000) was added and incubated. After
3,3',5,5'-tetramethylbenzidine (TMBZ, Sumitomo Bakelite, ML-1120T)
was added as a peroxidase substrate, enzyme activity was determined
by absorbance measurement at 450 nm. Purified human IgG antibody
having K chain (Sigma, I-3889) was used as standard. The standard
was diluted stepwise with mouse serum-supplemented PBS. In the case
of .mu. chain, 96-well microtiter plates were coated with
anti-human antibody .mu. chain mouse monoclonal antibody (Sigma,
I-6385) diluted with 50 mM carbonate-bicarbonate buffer (pH 9.6)
and then a serum sample was added. Subsequently, peroxidase-labeled
anti-human antibody .mu. chain mouse antibody (The Binding Site
Limited, MP008) was added to the plates and incubated. After TMBZ
(Sumitomo Bakelite, ML-1120T) was added, enzyme activity was
determined by absorbance measurement at 450 nm. Purified human IgM
antibody having .mu. chain (CAPPEL, 6001-1590) was used as
standard. The standard was diluted stepwise with mouse
serum-supplemented PBS. As a result, both the human antibody .mu.
and .kappa. chains were detected in both individuals. the
concentrations of these human antibodies in the sera increased
after the immunization (Tables 7 and 8).
10TABLE 7 Concentrations of Human Antibodies in Chimeric Mouse
KPG15 (ELISA) IgM (mg/l) Ig .kappa. (mg/l) Before Immunization 0.19
1.6 8 Days After Immunization 0.75 1.7
[0321]
11TABLE 8 Concentrations of Human Antibodies in Chimeric Mouse
KPG18 (ELISA) IgM (mg/l) Ig .kappa. (mg/l) Before Immunization 0.29
0.57 8 Days After Immunization 3.4 0.87
[0322] These results show that human antibody heavy and light chain
genes can function in the chimeric mice derived from the ES cells
retaining both human chromosome #2 partial fragments and human
chromosome #14 partial fragments.
EXAMPLE 21
[0323] Detection of Anti-HSA Human Antibody 7 Chain in sera of the
Human Chromosome #14 Fragments Transferred Chimeric Mice
[0324] The chimeric mice retaining human chromosome #14 fragments
which were produced by the same method as in Example 10 (K9 and
K11: both were derived from the TT2 cell clone 3-2, with chimerisms
of 50% and 30%, respectively) were immunized with HSA either 4
times at days 79, 93, 107 and 133 after birth (K9) or 3 times at
days 74, 88 and 111 after birth (K11) by the same method as in
Example 20. Antibodies including human .gamma. chain against human
serum albumin in the sera of the chimeric mice were detected by
ELISA. Ninety six-well microtiter plates were coated with HSA
(Sigma, A 3782) diluted with 50 mM carbonate-bicarbonate buffer (pH
9.6) and then a sample diluted with PBS was added. Subsequently,
peroxidase-labeled anti-human IgG mouse antibody (Pharmingen,
08007E) was added to the plates and incubated. After
O-phenylenediamine (OPD, Sumitomo Bakelite, ML-11300) was added as
a peroxidase substrate, enzyme activity was determined by
absorbance measurement at 490 nm. The titer of the anti-HSA human
IgG in the sera of the chimeric mice immunized with HSA increased
after the immunization. On the other hand, control ICR mouse gave a
background level of the anti-HSA human IgG titer after the
immunization with HSA. The results are shown in FIG. 17. In FIG.
17, the number of days after the first immunization of the chimeric
mice with HSA is plotted on the horizontal axis and the absorbance
at 490 nm is plotted on the vertical axis. These results show that
the antibody titer of the antigen specific human IgG was increased
by stimulation with the HSA antigen in the chimeric mice retaining
human chromosome #14 fragments.
EXAMPLE 22
[0325] Detection of Human Antibody .kappa. Chain in a Serum of the
Human Chromosome #22 Fragment Transferred Chimeric Mouse
[0326] The chimeric mouse K22-7 from Example 3 (9 weeks old; 10%
chimerism) was bled and human antibody .lambda. chain in the serum
was detected by ELISA (see Example 14). Ninety six-well microtiter
plates were coated with anti-human antibody .lambda. chain goat
antibody (VECTOR LABORATORIES INC., AI-3070) diluted with 50 mM
carbonate-bicarbonate buffer (pH 9.6) and then a serum sample was
added. Subsequently, biotin-labeled anti-human antibody.lambda.
chain goat antibody (VECTOR LABORATORIES INC., BA-3070) was added
to the plates and incubated. A complex of biotinylated horseradish
peroxidase and avidin DH (VECTOR LABORATORIES, INC., Vectastain ABC
Kit) was added and incubated. After TMBZ (Sumitomo Bakelite,
ML-1120T) was added as a peroxidase substrate, enzyme activity was
determined by absorbance measurement at 450 nm. Purified human IgG
antibody having .lambda. chain (Sigma, I-4014) was used as
standard. The standard was diluted stepwise with mouse
serum-supplemented PBS. As a result, human antibody A chain was
detected in the chimeric mouse at a concentration corresponding to
180 ng/ml of human IgG. These results show that human antibody
.lambda. chain gene can function in the chimeric mouse retaining a
human chromosome #22 fragment.
EXAMPLE 23
[0327] Detection of Human Antibody .kappa. Chain in sera of the
Human Chromosome #2 Fragment Transferred Chimeric Mice
[0328] The chimeric mouse K2-8 from Example 13 (5 weeks old; 70%
chimerism) and the chimeric mice K2-3, K2-4 and K2-12 from Example
13 (9 weeks old; chimerisms was 50%, 20% and 80%, respectively)
were bled and human antibody .kappa. chain in the sera was detected
by ELISA (see Example 14). Ninety six-well microtiter plates were
coated with anti-human antibody .kappa. chain goat antibody (VECTOR
LABORATORIES INC., AI-3060) diluted with 50 mM
carbonate-bicarbonate buffer (pH 9.6) and then a serum sample was
added. Subsequently, biotin-labeled anti-human antibody .kappa.
chain goat antibody (VECTOR LABORATORIES INC., BA-3060) was added
to the plates and incubated. A complex of biotinylated horseradish
peroxidase and avidin DH (VECTOR LABORATORIES, INC., Vectastain ABC
Kit) was added and incubated. After TMBZ (Sumitomo Bakelite,
ML-1120T) was added, enzyme activity was determined by absorbance
measurement at 450 nm. Purified human IgG antibody having .kappa.
chain (Sigma, I-3889) was used as standard. The standard was
diluted stepwise with mouse serum-supplemented PBS. The results are
shown in Table 9.
12TABLE 9 Concentration of Human Antibody .kappa. Chain in Chimeric
Mouse (ELISA) Chimeric Mouse Ig .kappa. (mg/l) K2-3 124 K2-4 85
K2-8 25 K2-12 56
[0329] The chimeric mice K2-3 and K2-4 retaining human chromosome
#2 fragments from Example 13 were immunized with HSA, 3 times at
days 66, 80 and 102 after birth by the same method as in Example
20. The chimeric mouse K2-12 was immunized with HSA, 4 times at
days 63, 77, 91 and 116 after birth by the same method as in
Example 20. Human antibody K chain against HSA in the sera of the
chimeric mice was detected by ELISA (see Example 14). Ninety
six-well microtiter plates were coated with HSA (Sigma, A 3782)
diluted with 50 mM carbonate-bicarbonate buffer (pH 9.6) and then a
sample was added. Subsequently, biotin-labeled anti-human antibody
K chain goat antibody (VECTOR LABORATORIES, INC., BA-3060) was
added to the plates and incubated. A complex of biotinylated
horseradish peroxidase and avidin DH (VECTOR LABORATORIES, INC.,
Vectastain ABC Kit) was added and incubated. After OPD (Sumitomo
Bakelite, ML-11300) was added as a peroxidase substrate, enzyme
activity was determined by absorbance measurement at 490 nm. The
titer of the anti-HSA human .kappa. chain in the sera of the
chimeric mice immunized with HSA increased after the immunization.
On the other hand, control ICR mouse gave a background level of the
anti-HSA human .kappa. chain titer after the immunization. The
results are shown in FIG. 18. In FIG. 18, the number of days after
the first immunization of the chimeric mice with HSA is plotted on
the horizontal axis and the absorbance at 490 nm is plotted on the
vertical axis. These results show that human antibody .kappa. chain
gene can function in the chimeric mice retaining human chromosome
#2 fragments and that the antibody titer of the antigen specific
human Ig .kappa. was increased by stimulation with the HSA antigen
in the chimeric mice.
EXAMPLE 24
[0330] Preparation of Human Antibody Heavy Chain (.mu. chain or
.gamma. chain)--Producing Hybridomas from the Human Chromosome #14
Transferred Chimeric Mouse
[0331] The spleen was removed from the HSA-immunized chimeric mouse
K9 (see Example 21) at day 136 after birth. A spleen cell was fused
with a myeloma cell to produce a hybridoma. The hybridoma was
produced using a myeloma cell Sp-2/0-Ag14 (Dainippon Pharmaceutical
Co., Ltd., 05-554) by the method described in Toyama and Ando,
"Monoclonal Antibody Experiment Procedure Manual", published by
Kodansha Scientific, 1991. The cells were inoculated into a medium
containing 10% ORIGEN Hybridoma Cloning Factor (HCF, Bokusui Brown)
in eight 96-well plates and G418 was added after 3 days at a
concentration of 1 mg/ml, followed by cultivation for 1-3 weeks.
The culture supernatant was analyzed by ELISA. Ninety six-well
microtiter plates were coated with anti-human .mu. chain mouse
monoclonal antibody (Sigma, I-6385) diluted with 50 mM
carbonate-bicarbonate buffer (pH 9.6) and a sample diluted with PBS
was added. Subsequently, peroxidase-labeled anti-human .mu. chain
mouse antibody (The Binding Site LIMITED, MP008) was added to the
plates and incubated.
2,2'-Azino-di-(3-ethyl-benzothiazoline-6-sulfonate) diammonium salt
(ABTS, Kirkegaard & Perry Laboratories Inc., 04-10-17) was used
as a substrate to detect seven positive clones. In the detection of
.gamma. chain-producing clones, 96-well microtiter plates were
coated with anti-human .gamma. chain mouse monoclonal antibody
(Sigma, I-6260) and a sample diluted with PBS was added.
Subsequently, peroxidase-labeled anti-human .gamma. chain mouse
antibody (Pharmingen, 08007E) was added to the plates and
incubated. ABTS (Kirkegaard & Perry Laboratories Inc.,
04-10-17) was used as a substrate and two human antibody .gamma.
chain-positive clones were obtained.
EXAMPLE 25
[0332] Preparation of Human Antibody Light Chain-Producing
Hybridomas from the Human Chromosome #2 Transferred Chimeric
Mouse
[0333] The spleen was removed from the HSA-immunized chimeric mouse
K2-3 (see Example 23) at day 105 after birth. A spleen cell was
fused with a myeloma cell to produce a hybridoma. The hybridoma was
produced using a myeloma cell P3.times.63Ag8.653 (Dainippon
Pharmaceutical Co., Ltd., 05-565) by the method described in Toyama
and Ando, "Monoclonal Antibody Experiment Procedure Manual",
published by Kodansha Scientific, 1991. The cells were inoculated
into a medium containing 10% HCF (Bokusui Brown) in ten 96-well
plates and G418 was added after 3 days at a concentration of 1
mg/ml, followed by cultivation for 1-3 weeks. The culture
supernatant was assayed by ELISA. The ELISA analysis was conducted
by the same method as in Example 23 and two human antibody .kappa.
chain-positive clones were obtained.
EXAMPLE 26
[0334] Re-Marking of the G418 Resistance-Marked Human Chromosome
#22 with Puromycin Resistance
[0335] The A9 cells retaining the G418 resistance-marked human
chromosome #22 (A9/#22 .gamma. 2) from Example 1 were re-marked
with puromycin resistance by the same method as in Example 16.
About 200 colonies of double-drug resistant clones obtained by
electroporation of the .gamma. 2 cells with pPGKPuro were collected
as one group and three such groups (P1, P2 and P3) were used as
donor cells to perform microcell transfer into wild-type mouse A9
cells. As a result, 6, 1 and 3 of double-drug resistant clones were
obtained from the groups P1, P2 and P3, respectively. The clone 6-1
from group P3 was used as a microcell donor cell and a wild-type A9
cell as a recipient cell to perform a microcell transfer experiment
(see Example 18). As a result, the human chromosome #22 was
confirmed to have been further marked with a puromycin resistance
gene. The preparation of microcells and the fusion with A9 cells
were conducted by the same methods as in Example 1. As a result,
twenty eight G418 resistant colonies appeared 11 days after the
microcell transfer. After the medium for these colonies was changed
to one containing 8 .mu.g/ml puromycin, these colonies were
cultured for 3 days to give 21 (75%) viable colonies. In many cases
of microcell fusion, only one or few chromosomes are transferred
into a recipient cell. Hence, cotransfer of both the resistance
genes at a high frequency shows that the G418 resistance-labeled
chromosome #22 retained in the 6-1 clone was marked with the
puromycin resistance gene.
EXAMPLE 27
[0336] Preparation and Sequencing of cDNA of a Human Antibody Heavy
Chain Variable Region from the Human Antibody Heavy Chain-Producing
Hybridoma
[0337] Among the human antibody heavy chain (IgM)-producing
hybridomas obtained in Example 15, H4B7 (HSA-specific) and H8F9
(non-specific) hybridomas were selected. Total RNAs were obtained
from these hybridomas using ISOGEN (Nippon Gene). The synthesis of
cDNA from 5 .mu.g each of the total RNAs was conducted with a
Ready-To-Go T-primed 1st strand Kit (Pharmacia Co.). Using the
resulting cDNA and the following primers prepared with reference to
Larrick et al., BIO/TECHNOLOGY, 7, 934-, 1989; Word et al., Int.
Immunol., 1, 296-, 1989, PCR was performed to amplify a human
antibody heavy chain variable region.
13 CM1 (human IgM constant region): 5'-TTGTATTTCCAGGAGAAAGTG (SEQ
ID NO:45) CM2 (ditto): 5'-GGAGACGAGGGGGAAAAGGG (SEQ ID NO:46) HS1
(human heavy chain variable region): 5'-ATGGACTGGACCTGGAGG(AG) (SEQ
ID NO:47) TC(CT)TCT(GT)C (a mixture of 8 sequences) HS2 (ditto):
5'-ATGGAG(CT)TTGGGCTGA(GC)CTGG(GC)TTT(CT)T (SEQ ID NO:48) (a
mixture of 16 sequences) HS3 (ditto):
5'-ATG(AG)A(AC)(AC)(AT)ACT(GT)TG(GT)(AT)(GCT)C(AT)(CT) (SEQ ID
NO:49) (GC)CT(CT)CTG (a mixture of 6144 sequences) * ( ) means that
any one of the bases therein should be selected.
[0338] In both cases of the H4B7 and H8F9 hybridomas, the first run
of PCR was performed by using three kinds of primer combinations of
HS1.times.CM1, HS2.times.CM1 and HS3.times.CM1 in 40 cycles at
94.degree. C. for 1 minute, 50.degree. C. for 2 minutes and
72.degree. C. for 3 minutes with a Thermal Cycler 140 (Perkin-Elmer
Corp.). The PCR products were amplified again under the same
temperature conditions in 30 cycles using HS1l.times.CM2,
HS2.times.CM2 and HS3.times.CM2 primers, respectively. The
amplification products were electrophoresed on a 1.5% agarose gel
and detected by staining with ethidium bromide. As a result, an
amplification product of about 490 bp was detected with the
HS3.times.CM2 primer in the case of the H4B7 hybridoma. In the case
of the H8F9 hybridoma, a slight band was detected at the same site
with the HS3.times.CM2 primer. The band in the case of H8F9 was
amplified again with the HS3.times.CM2 primer in 30 cycles under
the same temperature conditions as above. As a result, the
amplification product was detected as a very intensive signal.
These PCR products were cloned into a pBlueScriptII SK+(Stratagene
Ltd.) at a SmaI site in accordance with the method described in
Ishida et al., "Gene Expression Experiment Manual", published by
Kodansha Scientific, 1995. Among the amplification product-inserted
plasmids, plasmids #2, #3, #4(H4B7), #11, #13 and #14 (H8F9) were
selected and the nucleotide sequences of the amplification products
were determined with a Fluorescence Autosequencer (Applied
Biosystems Inc.). As a result of the comparison of the obtained
nucleotide sequences or deduced amino acid sequences with those of
known human antibody VH region (Marks et al., Eur. J. Immunol. 21,
985-, 1991) and JH region (Ravetch et al., Cell, 27, 583-, 1981),
it was revealed that both the H4B7 and H8F9 hybridomas contained a
combination of genes for VH4 family and JH2. These results show
that the chimeric mouse retaining human chromosome #14 partial
fragment produced a complete functional human antibody heavy chain
protein.
EXAMPLE 28
[0339] Preparation and Sequencing of cDNA of Human Antibody .kappa.
Chain from the Spleen of the Human Antibody .kappa.
Chain-Expressing Chimeric Mouse
[0340] In the same manner as in Example 5, cDNA was prepared from
the spleen of the chimeric mouse K2-8 from Example 13 which was
confirmed to express human antibody .kappa. chain in Example 23.
Using the resulting cDNA and the following primers prepared with
reference to Larrick et al. BIO/TECHNOLOGY, 7, 934-, 1989;
Whitehurst et al., Nucleic Acids Res., 20, 4929-, 1992, PCR was
performed to amplify human antibody .kappa. chain variable region.
cDNA from the liver of the chimeric mouse K2-8 and cDNA from the
spleen of the chimeric mouse K3-2-2 derived from the TT2/#14 3-2
clone (see Example 10) were used as negative controls.
14 KC2 (human Ig .kappa. chain constant region):
5'-CAGAGGCAGTTCCAGATTTC (SEQ ID NO:50) KC3 (ditto):
5'-TGGGATAGAAGTTATTCAGC (SEQ ID NO:51) KVMIX (human Ig .kappa.
chain variable region): 5'-ATGGACATG(AG)(AG)(AG)(AGT)(CT)C-
C(ACT)(ACG)G(CT)(GT)CA(CG)CTT (SEQ ID NO:52) (a mixture of 3456
sequences) * ( ) means that any one of the bases therein should be
selected.
[0341] PCR was performed by using primer combinations of
KVMIX.times.KC2 and KVMIX.times.KC3 in 40 cycles at 94.degree. C.
for 15 seconds, 55.degree. C. for 15 seconds and 72.degree. C. for
20 seconds with a Thermal Cycler 9600 (Perkin-Elmer Corp.). The
amplification products were electrophoresed on a 1.5% agarose gel
and detected by staining with ethidium bromide. As a result,
expected amplification products of about 420 bp (KC2) and about 450
bp (KC3) were detected. In the case of the two negative controls,
no specific amplification product was detected. These amplification
products were cloned into a pBlueScriptII SK+ (Stratagene Ltd.) at
a SmaI or EcoRI site in accordance with the method described in
Ishida et al., "Gene Expression Experiment Manual", published by
Kodansha Scientific, 1995. Among the amplification product-inserted
plasmids, VK-#1 clone derived from the KVMIX.times.KC2 primers was
selected and the nucleotide sequence of the amplification product
was determined with a Fluorescence Autosequencer (Applied
Biosystems Inc.). Since the obtained nucleotide sequence did not
contain a termination codon at any site between an initiation codon
and a constant region of human Ig.kappa. chain, the cloned
amplification products are believed to encode a variable region of
functional human Ig.kappa. chain. As a result of the comparison of
the obtained nucleotide sequences with those of known human
antibody V.kappa. region (Klein et al., Eur. J. Immunol. 23, 3248-,
1993) and J.kappa. region (Whitehurst et al., supra), it was
revealed that the VK-#1 clone contained a combination of genes for
V.kappa. 3 family and J.kappa. 4. These results show that the
chimeric mouse retaining human chromosome #2 partial fragment
produced a complete functional human antibody .kappa. chain
protein.
EXAMPLE 29
[0342] Detection and Quantitation of Human Antibody .gamma. Chain
Subclasses and .mu. Chain in sera of the Chimeric Mice Retaining
Human Chromosome #14 Fragment
[0343] The chimeric mice K15A and K16A from Example 10 (derived
from the 1-4 clone, with chimerism of 70% and 50%, respectively) of
11 weeks after birth were bled and human antibody .gamma. chain
subclasses and .mu. chain in the sera were detected by the same
ELISA method as in Example 14.
[0344] Quantative Determination of Human IgG1
[0345] Ninety six-well microtiter plates were coated with
anti-human IgG antibody (Sigma, I-6260) diluted with PBS. A serum
sample was added. Subsequently, peroxidase-labeled anti-human IgG1
antibody (Pharmingen, 08027E) was added to the plates and
incubated. After TMBZ (Sumitomo Bakelite, ML-1120T) was added,
enzyme activity was determined by absorbance measurement at 450 nm.
Purified human IgG1 antibody (Sigma, I-3889) was used as standard.
The standard was diluted stepwise with mouse serum-supplemented
PBS.
[0346] Quantative Determination of Human IgG2
[0347] Ninety six-well microtiter plates were coated with
anti-human IgG2 antibody (Sigma, I-9513) diluted with PBS. A serum
sample was added. Subsequently, peroxidase-labeled anti-human IgG
antibody (Sigma, A-0170) was added to the plates and incubated.
After TMBZ (Sumitomo Bakelite, ML-1120T) was added, enzyme activity
was determined by absorbance measurement at 450 nm. Purified human
IgG2 antibody (Sigma, I-4139) was used as standard. The standard
was diluted stepwise with mouse serum-supplemented PBS.
[0348] Quantative Determination of Human IgG3
[0349] Anti-human IgG3 antibody (Sigma, I-7260) was diluted with
100 mM glycine-HCl buffer (pH 2.5) and incubated for 5 minutes at
room temperature, followed by 10-fold dilution with 100 mM
phosphate buffer (pH 7.0). Ninety six-well microtiter plates were
coated with the anti-human IgG3 antibody solution. A serum sample
was added. Subsequently, peroxidase-labeled anti-human IgG antibody
(Pharmingen, 08007E) was added to the plates and incubated. After
TMBZ (Sumitomo Bakelite, ML-1120T) was added, enzyme activity was
determined by absorbance measurement at 450 nm. Purified human IgG3
antibody (Sigma, I-4389) was used as standard. The standard was
diluted stepwise with mouse serum-supplemented PBS.
[0350] Quantative Determination of Human IgG4
[0351] Anti-human IgG4 antibody (Sigma, I-7635) was diluted with
100 mM glycine-HCl buffer (pH 2.5) and incubated for 5 minutes at
room temperature, followed by 10-fold dilution with 100 mM
phosphate buffer (pH 7.0). Ninety six-well microtiter plates were
coated with the anti-human IgG3 antibody solution. A serum sample
was added. Subsequently, peroxidase-labeled anti-human IgG antibody
(Pharmingen, 08007E) was added to the plates and incubated. After
TMBZ (Sumitomo Bakelite, ML-1120T) was added, enzyme activity was
determined by absorbance measurement at 450 nm. Purified human IgG4
antibody (Sigma, I-4639) was used as standard. The standard was
diluted stepwise with mouse serum-supplemented PBS.
[0352] Quantative Determination of Human IgM
[0353] Ninety six-well microtiter plates were coated with
anti-human .mu. chain mouse monoclonal antibody (Sigma, I-6385)
diluted with PBS. A serum sample was added. Subsequently,
peroxidase-labeled anti-human .mu. chain mouse antibody (The
Binding Site Limited, MP008) diluted with mouse serum (Sigma,
M5905)-supplemented PBS was added to the plates and incubated.
After TMBZ (Sumitomo Bakelite, ML-1120T) was added as a peroxidase
substrate, enzyme activity was determined by absorbance measurement
at 450 nm. Purified human IgM having .mu. chain (CAPPEL, 6001-1590)
was used as standard. The standard was diluted stepwise with mouse
serum (Sigma, M5905)-supplemented PBS. The results are shown in
Table 10. All the subclasses IgG1, IgG2, IgG3 and IgG4, and IgM
were detected in the two chimeric mice K15A and K16A.
15TABLE 10 Concentrations of Human antibody IgG Subclasses and IgM
in the Chimeric Mice (ELISA) Chimeric mouse IgG1 IgG2 IgG3 IgG4 IgM
(mg/l) K15A 2.25 1.96 0.17 0.43 7.09 K16A 0.30 0.69 0.10 0.07
0.87
EXAMPLE 30
[0354] Preparation of Mouse ES Cell Clones (TT2) Retaining Human
Chromosome #22
[0355] The cell clone 6-1 (A9/#22, G418 and puromycin resistant)
from Example 26 was used as a chromosome donor cell for the
preparation of mouse ES cell (TT2) retaining human chromosome #22.
A wild-type TT2 cell line (see Example 9) was used as a chromosome
recipient cell. The microcell fusion and the selection of puromycin
resistant clones were conducted by the same procedures as in the
selection of G418 resistant clones in Example 9 except that the
concentration of puromycin was 0.75 .mu.g/ml. The frequency of the
appearance of the puromycin resistant clones was 1-2 per 10.sup.7
of TT2 cells. The puromycin resistant clones were stored frozen and
genomic DNA was prepared by the same methods as in Example 2. The
retention of human chromosome #22 in the puromycin resistant clone
PG22-1 was confirmed by the methods described in (1) and (2)
below.
[0356] (1) PCR Analysis
[0357] Genomic DNA of the puromycin resistant clone was used as a
template in PCR amplification. Among the genes on human chromosome
#22 (Genetic Maps, supra), ten primers whose presence in the A9/#22
clone was confirmed in Example 2 were used in the PCR
amplification. All the markers which existed in the A9/#22 clone
(see Example 2) were detected.
[0358] (2) Southern Blot Analysis
[0359] In accordance with the same method as described in Example 2
using human L1 sequence as a probe, Southern blot analysis was
conducted with genomic DNAs obtained from wild-type TT2 (negative
control), the chromosome donor cell 6-1 and the puromycin resistant
TT2 cell clone PG22-1. The results are shown in FIG. 19. In FIG.
19, the molecular weights of DNAs are shown at the left side. The
band pattern of the PG22-1 clone was equivalent to that of the 6-1
cell and the signal intensities were the same. Hence, it was
confirmed that chromosome #22 in the 6-1 cell had been transferred
certainly into the PG22-1 clone.
[0360] These experiments demonstrate that the puromycin resistant
TT2 cell clone PG22-1 retained the whole or the most part of human
chromosome #22.
EXAMPLE 31
[0361] Production of Chimeric Mice from the Mouse ES Cells (TT2)
Retaining Human Chromosome #22
[0362] The cells in a frozen stock of the puromycin resistant TT2
cell clone PG22-1 from Example 30 which was confirmed to retain
human chromosome #22 were thawed, started to culture and injected
into 8-cell stage embryos obtained by mating ICR or MCH(ICR) male
and female mice (CREA JAPAN, INC.); the injection rate was 10-12
cells per embryo. The embryos were cultured in a medium for ES
cells (see Example 9) overnight to develop to blastocysts. Two and
a half day after a foster mother ICR mouse was subjected to a
pseudopregnant treatment, about ten of the injected embryos were
transplanted to each side of the uterus of the foster mother. The
results are shown in Table 11.
16TABLE 11 Production of chimeric mice from the TT2 cell clone
retaining human chromosome #22 Number of ES ES cell Puromycin
cell-injected Number of Number Contribution clone/human resistant
8-cell stage offspring chimeric to coat color chromosome clone No.
embryos mice mice <20% 20-50% 50-80% TT2/#22 PG22-1 266 36 8 4 1
3
[0363] As a result of the transplantation of a total of 266
injected embryos, 36 offspring mice were born. Chimerism in the
offsprings can be determined by the extent of TT2 cell-derived
agouti coat color (dark brown) in the host embryo-derived albino
coat color. Out of the 36 produced offsprings, 8 mice were
recognized to have a partial agouti coat color, indicating the
contribution of the ES cells.
[0364] These results show that the ES cell clone (derived from TT2,
PG22-1) retaining human chromosome #22 maintain the ability to
produce chimera, that is, the ability to differentiate into normal
tissues of mouse.
EXAMPLE 32
[0365] Detection and Quantitation of Human Antibody .lambda. Chain
in sera of the Chimeric Mice Retaining Human Chromosome #22
[0366] The concentration of human antibody .lambda. in the sera of
the chimeric mice KPG22-1, 2 and 3 from Example 31 was determined
by ELISA in accordance with the same procedure as in Example 14.
The chimeric mice of 2 months after birth were bled and human
antibody A chain in the sera was detected by ELISA. Ninety six-well
microtiter plates were coated with anti-human immunoglobulin A
chain antibody (VECTOR LABORATORIES INC., IA-3070) diluted with PBS
and then a serum sample was added. Subsequently, biotin-labeled
anti-human immunoglobulin .lambda. chain antibody (VECTOR
LABORATORIES INC., BA-3070) was added to the plates and incubated.
A complex of biotinylated horseradish peroxidase and avidin DH
(VECTOR LABORATORIES, INC., Vectastain ABC Kit) was added and
incubated. After TMBZ (Sumitomo Bakelite, ML-1120T) was added,
enzyme activity was determined by absorbance measurement at 450 nm.
Purified human IgM antibody having .lambda. chain (Dainippon
Pharmaceutical Co., Ltd., U13200) was used as standard. The
standard was diluted stepwise with mouse serum-supplemented PBS.
The results are shown in Table 12. These results show that human
antibody .lambda. chain gene can function in the chimeric mice
retaining human chromosome #22.
17TABLE 12 Concentration of Human Antibody .lambda. Chain in
Chimeric Mice (ELISA) Chimeric Mouse % Chimerism Ig .lambda. (mg/l)
KPG22-1 50 12 KPG22-2 50 18 KPG22-3 20 24
EXAMPLE 33
[0367] Detection of Anti-Human HSA Human Antibody .lambda. Chain in
a Serum of the Human Chromosome #22 Transferred Chimeric Mouse
[0368] The chimeric mouse KPG22-3 from Example 31 was immunized
with HSA, 3 times at days 79, 94 and 110 after birth by the same
method as in Example 20. Human antibody .lambda. chain in the serum
of the chimeric mouse was detected by ELISA in accordance with the
same procedure as in Example 14. Ninety six-well microtiter plates
were coated with HSA (Sigma, A 3782) diluted with 50 mM
carbonate-bicarbonate buffer (pH 9.6) to a concentration of 5
.mu.g/ml and a serum sample was added. Biotinylated anti-human Ig
.lambda. antibody (VECTOR LABORATORIES INC., BA-3070) was added.
Subsequently, a complex of biotinylated-horseradish peroxidase and
avidin DH (VECTOR LABORATORIES, INC., Vectastain ABC Kit) was added
to the plates and incubated. After TMBZ (Sumitomo Bakelite,
ML-1120T) was added, enzyme activity was determined by absorbance
measurement at 450 nm. The titer of the anti-HSA human .lambda.
chain in the serum of the chimeric mouse increased after the
immunization. On the other hand, control ICR mouse gave a
background level of the anti-HSA human .lambda. chain titer after
the immunization with HSA. The results are shown in FIG. 20. In
FIG. 20, the number of days after the first immunization of the
chimeric mouse with HSA is plotted on the horizontal axis and the
absorbance at 450 nm is plotted on the vertical axis. These results
show that human antibody .lambda. chain gene can function in the
chimeric mouse retaining human chromosome #22 and that the antibody
titer of the antigen specific human Ig .lambda. was increased by
stimulation with the HSA antigen.
EXAMPLE 34
[0369] Preparation of Human Antibody Light Chain-Producing
Hybridomas from the Human Chromosome #22 Transferred Chimeric
Mouse
[0370] The spleen was removed from the mouse KPG22-3 (see Example
33) at day 113 after birth by the same method as in Example 25. A
spleen cell was fused with a myeloma cell to produce a hybridoma.
The hybridoma was produced using a myeloma cell SP-2/0-Ag14
(Dainippon Pharmaceutical Co., Ltd., 05-554) by the method
described in Toyama and Ando, "Monoclonal Antibody Experiment
Manual", published by Kodansha Scientific, 1991. The cells were
inoculated into a medium containing 10% HCF (Air Brown) in five
96-well plates and cultured for 1-3 weeks. The supernatant of the
culture solution in colony-positive wells was analyzed by ELISA.
The ELISA analysis was conducted by the same method as in Example
33 and four human antibody .lambda. chain-positive clones were
obtained.
EXAMPLE 35
[0371] Preparation of Mouse ES Cell Clones Retaining Both a Human
Chromosome #22 Partial Fragment and a Human Chromosome #14 Partial
Fragment
[0372] The 6-1 cell clone from Example 26 (A9/#22, G418 and
puromycin resistant) was used as a chromosome donor cell for the
preparation of mouse ES cells retaining both a human chromosome #22
partial fragment and a human chromosome #14 partial fragment. The
G418 resistant TT2 cell clone 1-4 retaining a human chromosome #14
partial fragment from Example 9 was used as a chromosome recipient
cell. The experiment of microcell fusion and the selection of
puromycin resistant cells were carried out by the same methods as
in the selection of the G418 resistant clones in Example 9 except
that the concentration of puromycin was 0.75 .mu.g/ml. As a result,
the frequency of the appearance of the puromycin resistant clones
was 1-2 per 10.sup.7 of 1-4 cells. The retention of G418 resistance
in the puromycin resistant clones was confirmed from the fact that
these clones were grown in the presence of 300 .mu.g/ml G418. The
double-drug resistant clones were stored frozen and genomic DNAs
were prepared by the same methods as in Example 2. The retention of
human chromosome #22 and a human chromosome #14 partial fragment in
the double-drug resistant clone PG22-5 was confirmed by PCR
analysis. With genomic DNA of the double-drug resistant clone used
as a template, PCR amplification was conducted using primers whose
presence on chromosome #22 was confirmed in Example 2 (A9/#22) and
primers whose presence on chromosome #14 was confirmed in Example 9
(TT2/#14 1-4); as a result, three markers (D22S275, D22S315 and Ig
.lambda.) of the ten markers on chromosome #22 and all of the
markers on chromosome #14 in the TT2/#14 1-4 clone were
detected.
[0373] These experiments demonstrate that the obtained double-drug
resistant TT2 cell clone retained both a human chromosome #22
partial fragment and a human chromosome #14 partial fragment.
EXAMPLE 36
[0374] Production of the Chimeric Mouse from the Mouse ES Cell
Clone Retaining Both a Human Chromosome #22 Partial Fragment and a
Human Chromosome #14 Partial Fragment
[0375] The cells in a frozen stock of the G418 and puromycin
double-resistant TT2 cell clone PG22-5 from Example 35 which was
confirmed to retain a human chromosome #22 partial fragment and a
human chromosome #14 partial fragment were thawed, started to
culture and injected into 8-cell stage embryos obtained by mating
ICR or MCH(ICR) male and female mice (CREA JAPAN, INC.); the
injection rate was 10-12 cells per embryo. The embryos were
cultured in a medium for ES cells (see Example 9) overnight to
develop to blastocysts. Two and a half day after a foster mother
ICR mouse was subjected to a pseudopregnant treatment, about ten of
the injected embryos were transplanted to each side of the uterus
of the foster mother. The results are shown in Table 13.
18TABLE 13 Production of the chimeric mouse from the mouse ES cell
clone retaining both a human chromosome #22 partial fragment and a
human chromosome #14 partial fragment Number of ES cell- ES cell
Double-drug injected Number of Number of Contribution clone/human
resistant 8-cell stage offspring chimeric to coat color chromosome
clone No. embryos mice mice <20% 20-50% 50-80% TT2/#22 + #14
PG22-5 302 16 5 3 2 0
[0376] As a result of the transplantation of a total of 302
injected embryos, 16 offspring mice were born. Chimerism in the
offsprings can be determined by the extent of TT2 cell-derived
agouti coat color (dark brown) in the host embryo-derived albino
coat color. Out of the 16 produced offsprings, 5 mice were
recognized to have a partial agouti coat color, indicating the
contribution of the ES cell.
[0377] These results show that the ES cell clone PG22-5 retaining a
human chromosome #22 partial fragment and a human chromosome #14
partial fragment maintains the ability to produce chimera, that is,
the ability to differentiate into normal tissues of mouse.
EXAMPLE 37
[0378] Detection of Human Antibody .lambda. Chain and .mu. Chain in
sera of the Chimeric Mice Derived from the ES Cells Retaining Both
a Human Chromosome #22 Partial Fragment and a Human Chromosome #14
Partial Fragment
[0379] The chimeric mice KPG22-9, 10 and 12 from Example 36 were
immunized with HSA. The chimeric mice KPG22-9 and 10 were immunized
11 weeks after birth and bled 2 weeks after the immunization. The
chimeric mouse KPG22-12 was immunized twice at 7 and 11 weeks after
birth and bled 2 weeks after the second immunization.
[0380] A serum human antibody .mu. chain, a serum human antibody
.lambda. chain, and a serum antibody having both human antibody
.lambda. and .mu. chains were detected by ELISA in accordance with
Example 14.
[0381] For the detection of complete human antibody molecules,
96-well microtiter plates were coated with anti-human
immunoglobulin .lambda. chain antibody (Kirkegaard & Perry
Laboratories Inc., 01-10-11) diluted with PBS and a serum sample
was added. Subsequently, peroxidase-labeled anti-human
immunoglobulin .mu. chain antibody (The Binding Site Limited,
MP008) was added to the plates and incubated. After TMBZ (Sumitomo
Bakelite, ML-1120T) was added as a peroxidase substrate, enzyme
activity was determined by absorbance measurement at 450 nm.
Purified human IgM antibody having .lambda. chain (Dainippon
Pharmaceutical Co., Ltd., U13200) was used as standard. The
standard was diluted stepwise with mouse serum-supplemented PBS.
Human antibody .mu. and .lambda. chains were detected and
determined quantitatively by ELISA in the same manner as in
Examples 29 and 32. The results are shown in Table 14.
19TABLE 14 Concentrations of Human Antibodies in Chimeric Mice
(ELISA) Chimerism ES clone Chimeric mouse (%) IgM (mg/l) Ig
.lambda. (mg/l) IgM, .lambda. (mg/l) PG22-5 KPG22-9 30 2.54 9.9
0.043 PG22-5 KPG22-10 5 4.96 21.5 0.333 PG22-5 KPG22-12 40 3.71 7.0
0.048 3-2 K9 50 6.66 -- <0.003 PG22-1 KPG22-2 50 -- 17.6
<0.003
[0382] Both .lambda. and .mu. chains were detected in the chimeric
mice. An antibody molecule having both human antibody .mu. and
.lambda. chains was detected. These results show: the human
antibody .lambda. chain gene and human antibody .mu. chain gene can
function at the same time in the chimeric mice derived from the ES
cells retaining human chromosome #22 partial fragments and human
chromosome #14 partial fragments; and a complete antibody
containing both human heavy and light chains was produced in part
of the B cells.
[0383] The control mice, that is, the chimeric mouse K9 retaining
only human chromosome #14 from Example 10 and the chimeric mouse
KG22-2 retaining only human chromosome #22 from Example 31, gave
background levels of an antibody having both human antibody
.lambda. and .mu. chains in the sera. It was confirmed that in
these detection systems, only a complete antibody molecule having
human .lambda. and .mu. chains was detected.
EXAMPLE 38
[0384] Detection of Human Antibody having Human .kappa. and .mu.
Chains in sera of the Chimeric Mice Derived from the ES Cells
Retaining Both Human Chromosome #2 Partial Fragments and Human
Chromosome #14 Partial Fragments
[0385] The chimeric mouse KPG-15 (derived from the TT2ES clone PG5,
10% chimerism) was immunized during 2-3 months after birth 3 times
with 0.2 ml of a solution of human serum albumin (HSA, Sigma,
A3782) and adjuvant (MPL+TDM Emulsion, RIBI Immunochem Research
Inc.) in PBS at a HSA concentration of 0.25 mg/ml and bled (see
Example 15). The chimeric mouse KPG-26 (derived from the TT2ES
clone PG6, 40% chimerism) of 6 weeks after birth was bled. The
concentration of a complete human antibody molecule in the sera was
determined by ELISA in accordance with Example 14. Ninety six-well
microtiter plates were coated with anti-human immunoglobulin
.kappa. chain antibody (Kirkegaard & Perry Laboratories Inc.,
01-10-10) diluted with PBS, and a serum sample was added.
Subsequently, peroxidase-labeled anti-human immunoglobulin .mu.
chain antibody (The Binding Site Limited, MP008) was added to the
plates and incubated. After TMBZ (Sumitomo Bakelite, ML-1120T) was
added as a peroxidase substrate, enzyme activity was determined by
absorbance measurement at 450 nm. Purified human IgM antibody
having .kappa. chain (CAPPEL, 6001-1590) was used as standard. The
standard was diluted stepwise with mouse serum-supplemented PBS.
The concentrations of .kappa. chain and .mu. chain were determined
by the same method as in Example 20. The results are shown in Table
15.
20TABLE 15 Concentrations of Human Antibodies in Chimeric Mice
(ELISA) Chimerism ES clone Chimeric mouse (%) IgM (mg/l) Ig .kappa.
(mg/l) IgM, .kappa. (mg/l) PG-5 KPG15 10 0.18 1.01 0.075 PG-6 KPG26
40 1.52 1.26 0.018 3-2 K9 50 6.66 -- <0.002 5-1 K2-9 40 -- 135
<0.002
[0386] A antibody molecule having both human antibody .mu. and
.kappa. chains was detected. The control mice, that is, the
chimeric mouse K9 retaining only human chromosome #14 from Example
10 and the chimeric mouse K2-9 retaining only human chromosome #2
from Example 13, gave background levels ( <0.002 mg/ml) of an
antibody having human antibody .kappa. and .mu. chains in the sera.
These results show: the human antibody .kappa. chain gene and human
antibody .mu. chain gene can function at the same time in the
chimeric mice derived from the ES cells retaining both human
chromosome #2 partial fragments and human chromosome #14 partial
fragments; and a complete antibody molecule containing both human
heavy and light chains was produced in part of the B cells.
EXAMPLE 39
[0387] Preparation of Mouse ES Cell Clone (TT2F, XO) Retaining a
Human Chromosome #2 Partial Fragment
[0388] The cell clone PG1 from Example 16 was used as a chromosome
donor cell for the preparation of a mouse ES cell (XO) retaining a
human chromosome #2 partial fragment. A TT2F cell (purchased from
Lifetec Oriental Co.) having a karyotype of (39, XO), which was
reported to differentiate efficiently into an oocyte in chimeric
mice (Shinichi Aizawa, "Biomanual Series 8, Gene Targeting"
published by Yodosha, 1995), was used as a chromosome recipient
cell. The experiment of microcell fusion and the selection of
puromycin resistant cells were carried out by the same methods as
in the selection of the G418 resistant clones in Example 9 except
that the concentration of puromycin was 0.75 .mu.g/ml. The
frequency of the appearance of the puromycin resistant clones was 5
per 10.sup.7 of TT2F cells. The puromycin resistant clones were
stored frozen and genomic DNAs were prepared from the clones by the
same methods as in Example 2. The retention of human chromosome #2
partial fragments in the drug resistant clones P-20 and P-21 was
confirmed by PCR analysis. As a result of PCR amplification using
genomic DNAs of the drug resistant clones as templates and three
kinds of primers C .kappa., FABP1 and V .kappa. 1-2 whose presence
in the A9/.andgate.2 W23 clone was confirmed in Example 1, all of
the three primers gave expected amplification products in both of
the two clones.
[0389] These experiments demonstrate that the obtained puromycin
resistant ES cell clone (TT2F, XO) retained a human chromosome #2
partial fragment.
EXAMPLE 40
[0390] Production of the Chimeric Mice from the Mouse ES Cell Clone
(TT2F, XO) Retaining a Human Chromosome #2 Partial Fragment
[0391] The cells in a frozen stock of the puromycin resistant TT2F
cell clone P-21 from Example 39 which was confirmed to retain a
human chromosome #2 partial fragment were thawed, started to
culture and injected into 8-cell stage embryos obtained by mating
ICR or MCH(ICR) male and female mice (CREA JAPAN, INC.); the
injection rate was 10-12 cells per embryo. The embryos were
cultured in a medium for ES cells (see Example 9) overnight to
develop to blastocysts. Two and a half day after a foster mother
ICR mouse was subjected to a pseudopregnant treatment, about ten of
the injected embryos were transplanted to each side of the uterus
of the foster mother. The results are shown in Table 16.
21TABLE 16 Production of the chimeric mice from the TT2F cell clone
retaining a human chromosome #2 partial fragment Number of ES cell-
ES cell Puromycin injected Number of Number of Contribution
clone/human resistant 8-cell stage offspring chimeric to coat color
chromosome clone No. embryos mice mice <20% 20-50% 50-90% 100%
TT2F/#2fg. P-21 141 20 9 0 2 3 4
[0392] As a result of the transplantation of a total of 141
injected embryos, 20 offspring mice were born. Chimerism in the
offsprings can be determined by the extent of TT2F cell-derived
agouti coat color (dark brown) in the host embryo-derived albino
coat color. Out of the 20 produced offsprings, 9 mice were
recognized to have a partial agouti coat color, indicating the
contribution of the ES cell. Four of the 9 mice were chimeric mice
having a full agouti coat color from the ES cells.
[0393] These results show that the ES cell clone P-21 retaining a
human chromosome #2 partial fragment maintains the ability to
produce chimera, that is, the ability to differentiate into normal
tissues of mouse.
EXAMPLE 41
[0394] Detection and Quantitative Determination of Human Antibody
.kappa. Chain in sera of the Chimeric Mice Derived from the TT2F
Clone Retaining a Human Chromosome #2 Partial Fragment
[0395] The chimeric mice K2-1F, 2F, 3F and 4F (derived from the
P-21 clone, 100% chimerism) from Example 40 of about 1 month after
birth were bled and the concentration of human antibody .kappa.
chain in the sera was determined quantitatively by ELISA in the
same manner as in Example 20.
[0396] The results are shown in Table 17. It was confirmed that the
human antibody .kappa. chain gene could function in the chimeric
mice when the TT2F was used as an ES cell.
22TABLE 17 Concentration of Human Antibody .kappa. Chain in
Chimeric Mice (ELISA) Chimeric mouse % Chimerism Ig .kappa. (mg/l)
K2-1F 100 66 K2-2F 100 156 K2-3F 100 99 K2-4F 100 20
EXAMPLE 42
[0397] Confirmation of the Retention of Human Chromosome in
Progenies of the Chimeric Mice Derived from the Mouse ES Cell
(TT2F, XO) Retaining a Human Chromosome #2 Partial Fragment
[0398] Examination was made as to whether ES cell-derived progenies
would be reproduced by mating the female chimeric mice K2-1F and
K2-4F (both were of 100% chimerism in coat color) from Example 40
with ICR male mice. In such a mating, offspring mice of an agouti
coat color should be reproduced from the oocytes derived from the
TT2F cell (agouti coat color, dominant) in the chimeric mice and
offspring mice of an albino coat color should be reproduced from
oocytes derived from ICR if the oocytes are fertilized with the
sperms from ICR male mice (albino, recessive). All the viable
offspring mice (K2-1F, 10 mice and K2-4F, 5 mice) obtained by one
mating of the respective combinations had an agouti coat color
which derived from the ES cells. The retention of human chromosome
fragments in genomic DNAs prepared from the tails of the offspring
mice was examined by a PCR method. As a result of the PCR
amplification using three kinds of primers whose presence in the
P-21 clone (see Example 39) was confirmed, the presence of these
three markers was confirmed in 4 out of the ten mice from K2-1F and
in 2 out of the five mice from K2-4F. The results of the PCR of
these 15 offspring mice are shown in FIG. 21. In FIG. 21, markers
(.PHI. X174/HaeIII fragment, Nippongene) and the DNA molecular
weights of main bands are shown at the right side and the lengths
of expected products of amplification with the respective primers
are shown by arrows at the left side. At the right side, the
results with tail-derived DNA of the mother chimeric mice K2-1F and
K2-4F (positive controls) are shown. These results show that the
TT2 cell clone P-21 differentiated into functional oocyte in the
chimeric mice and that a human chromosome #2 partial fragment was
transmitted to offsprings through oocyte.
EXAMPLE 43
[0399] Confirmation of the Retention of Human Chromosome in
Progenies of the Chimeric Mice Derived from the Mouse ES Cell (TT2,
XY) Retaining a Human Chromosome #2 Partial Fragment
[0400] Examination was made as to whether ES cell-contributed
offspring mice would be produced by mating K2-18 (70% chimeric male
mouse from Example 13) with K2-19 (60% chimeric female mouse of
Example 13) or non-chimeric female littermates. Since TT2 cell has
the karyotype of (40, XY), it may differentiate into a functional
sperm in the male chimeric mouse K2-18. If this is the case,
offspring mice of an agouti coat color should be reproduced from
ICR (albino, recessive)-derived oocytes fertilized with sperms from
TT2 cell (agouti color, dominant) in the chimeric mice. While a
total of 110 viable offspring mice were obtained by the mating, ten
had an agouti coat color which derived from the ES cells. The
retention of human chromosome fragments in genomic DNAs prepared
from the tails of 7 out of the ten offspring mice of an agouti coat
color was examined by a PCR method. As a result of PCR
amplification using two kinds of primers C .kappa. and FABP1 whose
presence in the 5-1 clone (TT2/.andgate.2fg. Example 12) was
confirmed and primer V .kappa. 1-2 which was shown in Example 1,
the presence of all of the three markers was confirmed in 2 out of
the seven mice. These results show that the TT2 cell clone 5-1
retaining a human chromosome #2 partial fragment differentiated
into functional sperms in the chimeric mice and that the human
chromosome #2 partial fragment was transmitted to offsprings
through the sperms.
EXAMPLE 44
[0401] Detection and Quantitative Determination of Human Antibody
.kappa. Chain in sera of Offspring Mice of the Chimeric Mice
[0402] The concentration of human antibody .kappa. chain in the
sera of the offspring mice K2-1F-1.about.10 and K2-4F-1.about.5
from Example 42 was determined quantitatively by ELISA. The mice of
about 4-6 months after birth were bled and the concentration of
human antibody .kappa. chain in the sera was determined by ELISA in
the same manner as in Example 20.
[0403] The results are shown in Table 18 together with the data
obtained in Example 42 on the retention of chromosome. It was
confirmed that the human antibody .kappa. chain gene can function
in the offspring mice reproduced from the chimeric mice.
23TABLE 18 Concentration of Human Antibody .kappa. Chain in
Offspring Mice (ELISA) Presence of Number of human chromosome #2
Mother mouse mouse fragments Ig .kappa. (mg/l) K2-1F #1 - 0.58
K2-1F #2 + 84.1 K2-1F #3 + 12.8 K2-1F #4 + 15.1 K2-1F #5 - 0.52
K2-1F #6 - 0.58 K2-1F #7 - 1.30 K2-1F #8 - 0.90 K2-1F #9 - 0.56
K2-1F #10 + 28.8 K2-4F #1 - <0.04 K2-4F #2 + 23.3 K2-4F #3 +
11.8 K2-4F #4 - 0.08 K2-4F #5 - 0.06
EXAMPLE 45
[0404] Analysis of Spleen Cells from the Human Chromosome #14
Partial Fragment Transferred Chimeric Mice
[0405] Flow cytometry analysis was accordance with the method
described in "New Biochemical Experiment Lecture 12, molecular
immunology I-Immunocells.cndot.Cytokines-", edited by the Japanese
Biochemical Society, 1989, published by Tokyo Kagaku Dojin; "Cell
Technology Separated Volume 8, New Cell Technology Experiment
Protocol", edited by the University of Tokyo, Medical Science
Institute, Anti-cancer Laboratory, 1991, published by Shujunsha;
and A. Doyle and J. B. Griffiths, "Cell & Tissue Culture:
Laboratory Procedures", published by John Wiley & Sons Ltd.,
1996. The spleen was removed from the chimeric mouse KPG06 (derived
from the PG16 clone, 30% chimerism) from Example 19 of six months
after birth and treated with an aqueous solution of ammonium
chloride. The spleen cells were stained with fluorescein
isothiocyanate (FITC)-labeled anti-mouse CD45R (B220) antibody
(Pharmingen, 01124A) in PBS containing 1% rat serum. After being
washed, the cells were reacted with 0.1 .mu.g of biotin-labeled
anti-human IgM antibody (Pharmingen, 08072D) or a control
biotin-labeled anti-human .lambda. chain antibody (Pharmingen,
08152D) in PBS containing 5% mouse serum and stained with 0.1 .mu.g
of streptoavidin-phycoerythrin (Pharmingen, 13025D), followed by
analysis with a flowcytometer (Becton Dickinson Immunocytometry
Systems, FACSort). An ICR mouse retaining no human chromosome was
used as a control for analysis by the same method. The results are
shown in FIG. 22. In FIG. 22, the human IgM is plotted on the
horizontal axis and the CD45R (B220) is plotted on the vertical
axis. A population of cells positive to both B cell marker CD45R
(FITC) and human IgM (PE) increased by 4%, indicating that cells
expressing human antibody .mu. chain on the cell surfaces were
present in the chimeric mice.
EXAMPLE 46
[0406] Cloning and Sequencing of Variable Regions of Human Antibody
Genes from cDNA Derived from the Spleen of the Chimeric Mice
Expressing Human Antibody Heavy Chain, .kappa. and .lambda. Chains,
Respectively
[0407] In the same manner as in Example 5, cDNAs were synthesized
from RNAs extracted from the spleens of the chimeric mice K15A
(derived from the 1-4 clone, prepared by the method described in
Example 10), K2-8 prepared in Example 13 and KPG22-2 prepared in
Example 31, all of which were confirmed to express human antibody
heavy chain, .kappa. and .lambda. chains in Examples 29, 23 and 32,
respectively. PCR was performed using the respective cDNAs and the
following primers to amplify the variable regions of respective
human antibody. cDNA derived from the spleen of a non-chimeric
mouse ICR was used as a negative control. Those primers set forth
below without indication of reference literature were designed on
the basis of the nucleotide sequences obtained from data bases such
as Genebank and the like.
[0408] K15A (Heavy Chain)
[0409] For constant region:
24 HIGMEX1-2: 5'-CCAAGCTTCAGGAGAAAGTGATGGAGTC (SEQ ID NO:53)
HIGMEX1-1: 5'-CCAAGCTTAGGCAGCCAACGGCCACGCT (used in 2nd PCR of (SEQ
ID NO:54) VH3BACK)
[0410] For variable region:
[0411] VH1/5BACK (59.degree. C., 35 cycles, Marks et al., Eur. J.
Immnol., 21, 985-, 1991),
[0412] VH4BACK (59.degree. C., 35 cycles, Marks et al., supra),
and
[0413] VH3BACK (1st PCR:59.degree. C., 35 cycles; 2nd
PCR:59.degree. C. , 35 cycles, Marks et al., supra)
[0414] K2-8 (Light Chain .kappa.)
[0415] For constant region:
[0416] KC2H: 5'-CCAAGCTTCAGAGGCAGTTCCAGATTTC (SEQ ID NO: 55)
[0417] For variable region:
[0418] Vk1/4BACK (55.degree. C., 40 cycles, Marks et al., Eur. J.
Immnol., 21, 985-, 1991),
[0419] Vk2BACK (55.degree. C., 40 cycles, Marks et al., supra),
and
[0420] Vk3BACK (55.degree. C., 40 cycles, Marks et al., supra)
[0421] KPG22-2 (Light Chain .lambda.)
[0422] For constant region:
[0423] C.lambda. MIX (a mixture of the following three kinds of
primers at an equal molar ratio)
25 IGL1-CR: 5'-GGGAATTCGGGTAGAAGTCACTGATCAG (SEQ ID NO:56) IGL2-CR:
5'-GGGAATTCGGGTAGAAGTCACTTATGAG (SEQ ID NO:57) IGL7-CR:
5'-GGGAATTCGGGTAGAAGTCACTTACGAG (SEQ ID NO:58)
[0424] For variable region:
[0425] V.lambda. 1LEA1 (55.degree. C., 40 cycles, Williams et al.,
Eur. J. Immunol., 23, 1456-, 1993),
[0426] V.lambda. 2MIX (55.degree. C., 40 cycles, a mixture of
V.lambda. 2 LEAL and V.lambda. 2 JLEAD (Williams et al. (supra)) at
an equal molar ratio)
[0427] V.lambda. 3MIX (55.degree. C., 40 cycles, a mixture of
V.lambda. 3LEA1, V.lambda. 3JLEAD and V.lambda. 3BACK4, which were
reported in Williams et al. (supra) at an equal molar ratio.
[0428] The PCR was performed with combinations of the primers for
constant regions with those for variable regions (3 primer pairs
each for heavy chain, .kappa. and .lambda. chains) at 94.degree. C.
for 15 seconds, at the annealing temperatures shown with respect to
the respective primers for variable region for 15 seconds, at
72.degree. C. for 20 seconds in the cycle numbers shown with
respect to the respective primers for variable region using a
Thermal Cycler 9600 (Perkin-Elmer Corp.). In the second run of PCR
using VH3BACK, the amplification products of the first run of PCR
were amplified again with a combination of the two primers
H1GMEX1-1 and VH3BACK. All of the amplification products were
electrophoresed on a 1.5% agarose gel and stained with ethiduim
bromide for detection. As a result, the amplification products
having expected lengths (heavy chain, about 470 bp; light chain
.kappa., about 400 bp; and light chain .lambda., about 510 bp) were
detected in all of the combinations. In the negative control,
specific amplification product was not detected at the same
position in any of the combinations. The obtained amplification
products were extracted from the agarose gel using prep.A.gene
(Bio-Rad Laboratories, Inc.), treated with restriction enzymes
(heavy chain, HindIII and PstI; light chain .kappa., HindIII and
PvuII; and light chain .lambda., HindIII and EcoRI), and cloned
into pUC119 (TAKARA SHUZO CO., LTD.) at the sites of HindIII/PstI
(heavy chain), HindIII/HincII (.kappa. chain) and HindIII/EcORI
(.lambda. chain). The nucleotide sequences of the products that
were amplified with the following primers and which were cloned
into the plasmids were determined with a Fluorescence Autosequencer
(Applied Biosystems Inc.).
[0429] HIGMEX1-2.times.VH1/5BACK: 10 clones
[0430] HIGMEX1-2.times.VH4BACK: 8 clones
[0431] HIGMEX1-2 (2nd PCR, HIGMEX1-1).times.VH3BACK: 5 clones
[0432] KC2H.times.V .kappa. 1/4BACK: 6 clones
[0433] KC2H.times.V .kappa. 2BACK: 7 clones
[0434] KC2H.times.V .kappa. 3BACK: 4 clones
[0435] C.lambda. MIX.times.V.lambda. 1LEA1: 5 clones
[0436] C.lambda. MIX.times.V.lambda. 2MIX: 6 clones
[0437] C.lambda. MIX.times.V.lambda. 3MIX: 5 clones
[0438] The obtained nucleotide sequences were analyzed with DNASIS
(Hitachi Software Engineering Co., Ltd.). The results show that all
of the sequences were derived from human and that they were
functional sequences which did not contain a termination codon at
any site between an initiation codon and a constant region: this
was true with all of the .kappa. and .lambda. chains and with 21
out of a total of 23 heavy chains. When the same sequences were
removed from the determined sequences, unique variable region
sequences were identified as follows: 17 heavy chains, 11 .kappa.
chains, and 12 .lambda. chains.
EXAMPLE 47
[0439] Analysis of the Nucleotide Sequences of Variable Region of
Human Antibody Genes from cDNA Derived from the Spleen of the
Chimeric Mouse Expressing Human Antibody Heavy Chain, .kappa. and
.lambda. Chains, Respectively
[0440] The nucleotide sequences determined in Example 46 (heavy
chain, 17 clones; .kappa. chain, 11 clones; and .lambda. chain, 12
clones) were analyzed in the following points.
[0441] 1. Identification of known germ line V gene segments used in
the respective variable regions
[0442] 2. Identification of known germ line J gene segments used in
the respective variable regions
[0443] 3. Identification of known germ line D gene segments used in
the heavy chain variable regions
[0444] 4. Identification of the addition of N region in the heavy
chain variable regions on the basis of the results of 1, 2 and
3
[0445] 5. Determination of the amino acid sequences deduced from
the nucleotide sequences of the respective variable regions
[0446] The results are shown in Table 19. For the identification in
points of 1 and 2, search for homology with germ line V and J
segments registered in Genbank and the like was conducted with
DNASIS. The VH segments, V .kappa. segments and V.lambda. segments
are shown in Table 19 together with the family names of the
respective V fragments in accordance with the conventions described
in Cook et al., Nature genetics, 7, 162-, 1994 (VH fragments),
Klein et al., Eur. J. Immunol, 23, 3248-, 1993 (V .kappa.
fragments) and Williams et al. (supra) (V.lambda.0 fragments),
respectively. For the identification in point 3, search for
homology with germ line D fragments reported in Ichihara et al.,
The EMBO J., 7, 13, 4141-, 1988 was conducted with DNASIS.
Assignment was based on at least 8 bp identity and the results are
shown in Table 19. DN1 is believed to be the new DN family segment
reported in Green et al., Nature Genetics, 7, 13-, 1994. For the
identification in point 4, the nucleotide sequences which did not
appear in any germline sequences were determined to be N regions on
the basis of the results for 1(V), 2(J) and 3 (D). As a result, N
region was observed in 11 of the 13 sequences in which D segment
was identified and its average length was 8.7 bp. For the
determination in point 5, the respective sequences were converted
by DNASIS to amino acid sequences which were expressed with one
letter symbols. In Table 19, only CDR3 region is shown. At the
right side of Table 19, the names of the primers used in cloning of
the respective variable regions and the names of clones are
shown.
26TABLE 19 V family V segment CDR3 J (D) V primer Clone K15A VH1
VH1-8 VRSSSWYEYYYYGMDV J6 (DN1) VH4BACK H4-10 VH1-18
GGITMVRGLIITDWYFDL J2 (DXP'1) VH1/5BACK H1-7 VH1-24 APYSGRFDY J4
(DK1) VH1/5BACK H1-6 VH1-46 ERYYGSGSYQDYYYYYGMDV J6 (DXP'1)
VH1/5BACK H1-2 VH1-46 GGYSGYEDYYYYGMDV J6 (DK1) VH1/5BACK H1-10 VH2
VH2-5 SYFDWPDFDY J4 (DXP1) VH4BACK H4-14 VH3 VH3-21
EGCSGGSCLPGYYYYGMDV J6 (DLR2) VH1/5BACK H1-4 VH3-23 AHGDPYFDY J4
VH1/5BACK H1-3 VH3-23 DADAFDI J3 VH1/5BACK H1-8 VH3-23 SGWDY J4
(DN1*) VH3BACK H3-3 VH3-23 TGFDL J2 VH4BACK H4-4 VH3-33
EGGYGSVGDYYYYGMDV J6 (DXP'1) VH1/5BACK H1-9 VH3-33 GGYSYGYDYYYYGMDV
J6 (DXP'1) VH3BACK H3-5 VH3-33 GYSSGWYDY J4 (DN1*) VH4BACK H4-9 VH4
VH4-34 RYSSGWYYFDY J4 (DN1*) VH4BACK H4-15 VH4-59 GRIAVASFDY J4
(DN1*) VH4BACK H4-2 VH4-59 GSGSYFHFDY J4 VH4BACK H4-6 K2-8 V
.kappa. 1 O18-8 QQHDNLPFT J3 V .kappa. 1BACK K1-1 O18-8 QQYDNLPIT
J5 V .kappa. 1BACK K1-3 O18-8 QQHDNLPFA J3 V .kappa. 2BACK K2-2 L1
QQYNSYPLT J4 V .kappa. 1BACK K1-6 V .kappa. 2 A17 MQGTHLLT J4 V
.kappa. 2BACK K2-1 A17 MQGTHWIT J5 V .kappa. 2BACK K2-5 V .kappa. 3
A27 QQYGSSPTWT J1 V .kappa. 3BACK K3-1 A27 QQYGSSPFT J3 V .kappa.
3BACK K3-4 A27 QQYGSSPLWT J1 V .kappa. 3BACK K3-5 A27 QQYGSSPPWT J1
V .kappa. 3BACK K3-6 V .kappa. 6 A26-10 HQSSSLPQT J1 V .kappa.
2BACK K2-4 KPG22-2 V .lambda. 1 DPL3 AAWDDSLDVV JC3 V .lambda.
1LEA1 L1-3 DPL5 GTWDSSLSAGV JC2 V .lambda. 1LEA1 L1-4 DPL5
GTWDSSLSAGVV JC3 V .lambda. 1LEA1 L1-6 DPL5 GTWDSSLSAVV JC2 V
.lambda. 1LEA1 L1-9 DPL8 QSYDSSLSGVV JC3 V .lambda. 1LEA1 L1-8 V
.lambda. 2 DPL10 CSYAGSSTLV JC2 V .lambda. 2MIX L2-4 DPL11
SSYTSSSTVV JC2 V .lambda. 2MIX L2-1 DPL11 SSYTSSSTLV JC2 V .lambda.
2MIX L2-3 DPL11 CSYTSSSTFV JC2 V .lambda. 2MIX L2-7 DPL12
SSYAGSNNLV JC3 V .lambda. 2MIX L2-5 DPL12 SSYAGSNNFVV JC3 V
.lambda. 2MIX L2-6 V .lambda. 3 DPL16 NSRDSSGNLV JC2 V .lambda.
3MIX L3-1
EXAMPLE 48
[0447] Preparation of a Targeting Vector for Knocking Out Antibody
Genes (Heavy-Chain and Light-Chain .kappa. Genes) in TT2 (or TT2F)
ES Cells
[0448] It becomes possible to transfer a human chromosome #14
fragment marked with a G418 resistance gene (Example 9) and human
chromosome #2 (Example 18) or #22 (Example 35) marked with a
puromycin resistance gene into TT2 (or TT2F) cells in which mouse
antibody genes (heavy-chain, light-chain .kappa.) are disrupted.
Those chimeric mice which are produced from these human chromosomes
#14+#2 or #14+#22-transferred, mouse antibody genes (heavy-chain,
light-chain .kappa.)-disrupted TT2 (or TT2F) ES cells according to
the method of Example 19 (heavy-chain+.kappa. chain) or Example 36
(heavy-chain+.lambda. chain) are expected to produce antibodies
both heavy- and light-chains of which are mainly derived from
humans. The abbreviations of the restriction enzymes, etc.
appearing in FIGS. 23-27 are as follows.
[0449] Restriction enzymes: Kp: KpnI, B: BamHI, RI: EcoRI, RV:
EcoRV, N: NotI, SII: ScaII, Sca: ScaI, Sfi: SfiI, Sm: SmaI, X:
XhoI, SI: SalI, dKp: deletion of KpnI, (X): XhoI restriction site
from .lambda. vector
[0450] Dotted portion: pBluescript SKII(+) plasmid DNA
[0451] : LoxP sequence
[0452] 1. Preparation of Plasmid LoxP-pstNEO in Which LoxP Sequence
is Inserted at Both the Ends of a G418 Resistance Gene
[0453] For the deletion of a G418 resistance gene after knocking
out an antibody gene of TT2 or (TT2F) cells, it is necessary to
insert LoxP sequence (Sauer et al., Proc. Natl. Acad. Sci. USA, 85,
5166-, 1988) which is the recognition sequence of Cre recombinase
(Sauer et al., supra) at both the ends of the G418 resistance gene
(Example 1) in the same direction. Briefly, pstNEO gene was cut out
from pSTneoB plasmid DNA (Example 1) with restriction enzyme XhoI.
The DNA fragment was purified by agarose gel electrophoresis and
then blunted with T4-DNA polymerase (Blunting End Kit from Takara
Shuzo). LoxP sequence-containing plasmid DNA pBS246 (Plasmid
pBS246, loxP2 Cassette Vector, U.S. Pat. No 4,959,317) was
purchased from GIBCO BRL. XhoI linker DNAs were inserted into the
EcoRI and SpeI restriction sites of this plasmid. The pstNEO DNA
fragment described above was inserted into the EcoRV restriction
site of the thus modified pBS246 to give plasmid LoxP-pstNEO (FIG.
23).
[0454] 2. Isolation of Genomic DNA Clones Containing
C57BL/6-Derived Antibody Heavy-Chain C .mu. (IgM Constant Region)
or Light-Chain J.kappa.-C.kappa. (Ig.kappa. Joint Region and
Constant Region)
[0455] Since TT2 (or TT2F) cells were derived from F1 mice between
C56BL/6 mice and CBA mice, the inventors have decided to prepare
vectors for antibody gene knockout using genomic DNA clones derived
from a C57BL/6 mouse. As a genomic DNA library, an adult C57BL/6N
male liver-derived .lambda. DNA library from Clontech was used. As
a probe for screening, the following synthetic DNA sequences (60
mers) were used.
27 Heavy-chain C.mu. probe: 5'-ACC TTC ATC GTC CTC TTC CTC CTG AGC
CTC TTC (SEQ ID NO:59) TAC AGC ACC ACC GTC ACC CTG TTC AAG-3'
Light-chain .kappa. probe: 5'-TGA TGC TGC ACC AAC TGT ATC CAT CTT
CCC ACC ATC (SEQ ID NO:60) CAG TGA GCA GTT AAC ATC TGG AGG-3'
[0456] The .lambda. clones were isolated and analyzed to subclone
those DNA fragments containing heavy-chain C .mu. or light-chain
J.kappa.-C.kappa. into plasmid pBluescript SKII(+) (Stratagene)
(heavy-chain C.mu.: FIG. 24; light-chain J.kappa.-C.kappa.: FIG.
25). These DNA fragments were used to prepare targeting vectors for
disrupting mouse antibody genes in TT2 (or TT2F) cells as described
below.
[0457] 3. Preparation of a Vector Plasmid for Disrupting a Mouse
Antibody Heavy-Chain Gene
[0458] In the C.mu.-encoding region in the genomic DNA fragment
containing a mouse antibody heavy-chain constant region which was
prepared in 2 above, a DNA fragment containing the 2nd to 4th exons
(BamHI-XhoI) was replaced with the LoxP-pstNEO gene prepared in 1
above (FIG. 26). The direction of transcription of pstNEO was
opposite to the direction of transcription of the antibody gene.
This plasmid DNA was amplified using E. coli JM109 and purified by
cesium chloride equilibrium centrifugation ("Introduction to Cell
Technology Experimental operations", published by Kodansha, 1992).
The purified plasmid DNA was cleaved at one site with restriction
enzyme SacII and used for transfection of TT2 (or TT2F) ES cells.
As a probe for Southern blot analysis of transformant genomic DNA
to detect from transformant TT2 (or TT2F) ES cells those clones in
which homologous recombination has taken place in the antibody
heavy-chain portion with the targeting vector, a DNA fragment
(about 500 bp) of the switch region located upstream of C
.mu.-encoding region. This DNA fragment was obtained by amplifying
129 mouse genomic DNAs by PCR under the following conditions.
28 Sense primer: 5'-CTG GGG TGA GCC GGA TGT TTT G-3' (SEQ ID NO:
61) Antisense primer: 5'-CCA ACC CAG CTC AGC CCA GTT C-3' (SEQ ID
NO: 62)
[0459] Template DNA: 1 .mu.g of EcoRI-digested 129 mouse genomic
DNAs The reaction buffer, deoxynucleotide mix and Taq DNA
polymerase used were from Takara Shuzo.
[0460] Reaction conditions: 94.degree. C., 3 min, 1
cycle.fwdarw.94.degree. C., 1 min; 55.degree. C., 2 min; 72.degree.
C., 2 min; 3 cycles.fwdarw.94.degree. C., 45 sec; 55.degree. C., 1
min; 72.degree. C., 1 min; 36 cycles
[0461] After it was confirmed that amplified DNA fragment can be
cleaved at one site with restriction enzyme HindIII as indicated in
the Genbank database, this DNA fragment was subcloned into the
EcoRV restriction site of plasmid pBluescript. This plasmid DNA
(S8) was digested with restriction enzymes BamHI and XhoI. A PCR
fragment (about 550 bp) was purified by agarose gel electrophoresis
to give a probe. Genomic DNA from those TT2 (or TT2F) ES cells
transformed with the targeting vector is digested with restriction
enzymes EcoRI and XhoI, and separated by agarose gel
electrophoresis. Then, Southern blotting is performed using the
above probe.
[0462] 4. Preparation of a Vector for Disrupting the Mouse Antibody
Light-Chain .kappa. Gene
[0463] The genomic DNA fragment prepared in 2 above contains the J
region and constant region of mouse antibody light-chain .kappa.. A
DNA fragment (EcoRI-SacII) containing the J region (J1-J5) was
replaced with the LoxP-pstNEO gene prepared in 1 above (FIG. 27).
The direction of transcription of pstNEO was the same as that of
the antibody gene. This plasmid DNA was amplified using E. coli
JM109 and purified by cesium chloride equilibrium centrifugation.
The purified plasmid DNA was cleaved at one site with restriction
enzyme KpnI and used for transfection of TT2 (or TT2F) ES cells. As
a probe for Southern blot analysis of transformant genomic DNA to
detect from transformant TT2 (or TT2F) ES cells those clones in
which homologous recombination has taken place in the antibody
heavy-chain portion with the targeting vector a DNA fragment at the
3' of the light-chain J.kappa.-C.kappa. genomic DNA fragment (see
FIG. 25) (XhoI-EcoRI; about 1.4 kbp) was used. Genomic DNA from
those TT2 (or TT2F) ES cells transformed with the targeting vector
is digested with restriction enzymes EcoRI and NotI, and separated
by agarose gel electrophoresis. Then, Southern blotting is
performed using the above probe.
EXAMPLE 49
[0464] Production of a Mouse ES Cell Antibody Heavy-Chain
Gene-Disrupted Clone
[0465] In order to obtain a recombinant in which an antibody
heavy-chain gene has been disrupted by homologous recombination
(hereinafter, referred to as an "antibody heavy-chain homologous
recombinant"), the antibody heavy-chain targeting vector prepared
in Section 3, Example 48 was linearized with restriction
enzyme-SacII (Takara Shuzo), and transferred into mouse TT2F ES
cells according to the method described by Shinichi Aizawa,
"Biomanual Series 8, Gene Targeting", published by Yodosha, 1995.
The TT2F cells were treated with trypsin and suspended in HBS at a
concentration of 2.5.times.10.sup.7 cells/ml. To the cell
suspension, 5 .mu.g of DNA was added. Then, electroporation was
performed with a gene pulser (Bio-Rad Laboratories, Inc.; resistor
unit not connected). A voltage of 250 V was applied at a
capacitance of 960 .mu.F using an electroporation cell of 4 mm in
length at room temperature. The electroporated cells were suspended
in 20 ml of an ES medium and inoculated into two tissue culture
plastic plates (Corning) of 100 mm into which feeder cells were
seeded preliminarily. Similarly, experiments using 10 and 15 .mu.g
of DNA were also conducted. After one day, the medium was replaced
with a medium containing 300 .mu.g/ml of G418 (GENETICIN; Sigma).
Seven to nine days thereafter, a total of 176 colonies formed were
picked up. Each colony was grown up to confluence in a 12-well
plate, and then four fifths of the culture was suspended in 0.2 ml
of a preservation medium [ES medium+10% DMSO (Sigma)] and stored
frozen at -80.degree. C. The remaining one fifth was inoculated
into a 12-well gelatin coated plate and cultured for 2 days. Then,
genomic DNA was obtained by the method described in Example 2.
These genomic DNAs from G418 resistant TT2F cells were digested
with restriction enzymes EcoRI and XhoI (Takara Shuzo) and
separated by agarose gel electrophoresis. Then, Southern blotting
was performed to detect homologous recombinants with the probe
described in Section 3, Example 48. As a result, 3 clones out of
the 176. clones were homologous recombinants. The results of
Southern blot analysis of wild-type TT2F cells and homologous
recombinants #131 and #141 are shown in the left-side three lanes
in FIG. 28. In wild-type TT2F cells, two bands (a and b) are
detected which were obtained by the EcoRI and XhoI digestion. In
the homologous recombinants, it is expected that one of these bands
disappears and that a new band (c) will appear at the lower part of
the lane. Actually, band (a) has disappeared in #131 and #141 in
FIG. 28 and a new band (c) has appeared. The size of DNA is shown
at the left side of the Figure. These results show that one allele
of an antibody heavy-chain gene in these recombinant clones has
been disrupted by homologous recombination.
EXAMPLE 50
[0466] Production of Chimeric Mice from Antibody Heavy-Chain
Homologous Recombinant ES Cells
[0467] The cells in a frozen stock-of the antibody heavy-chain
homologous recombinant TT2F cell clone #131 from Example 49 were
thawed, started to culture and injected into 8-cell stage embryos
obtained by mating a male and a female mouse of ICR or MCH(ICR)
(CREA JAPAN, INC.); the injection rate was 10-12 cells per embryo.
After the embryos were cultured overnight in the medium for ES
cells (see Example 9) to develop into blastocysts, about ten of the
TT2F cell-injected embryos were transplanted to each side of the
uterus of a foster mother ICR mouse (CREA JAPAN, INC.; 2.5 days
after pseudopregnant treatment). As a result of transplantation of
a total of 94 injected embryos, 22 offspring mice were born.
Chimerism in the offsprings can be determined by the extent of TT2F
cell-derived agouti coat color (dark brown) in the host embryo
(ICR)-derived albino coat color (white). Out of the 22 offsprings,
18 mice were recognized to have partial agouti coat color,
indicating the contribution of the ES cells. Out of the 18 mice, 16
mice were female chimeric mice in which more than 80% of their coat
color was agouti (i.e. ES cell-derived). From these results, it was
confirmed that the antibody heavy-chain homologous recombinant ES
cell clone #131 retains the ability to produce chimera. Since a
large number of the resultant chimeric mice are female mice
exhibiting extremely high contribution, it is very likely that the
ES cells have differentiated into functional germ cells (oocytes).
Two female chimeric mice exhibiting 100% contribution were mated
with MCH(ICR) male mice. As a result, all of the offspring mice
exhibited agouti coat color. These offsprings are derived from #131
(see Example 42), and thus it is considered that a disrupted
antibody heavy-chain allele was transmitted to them at a rate of
50%.
EXAMPLE 51
[0468] Production of a Double Knockout Clone from the Antibody
Heavy-Chain Homologous Recombinant
[0469] It has been reported that a clone in which both alleles are
disrupted can be obtained by disrupting one allele by insertion of
a G418 resistance gene, culturing an ES cell clone in a medium with
an increased G418 concentration and screening the resultant high
concentration G418 resistant clones (Shinichi Aizawa, "Biomanual
Series 8, Gene Targeting", published by Yodosha, 1995). Based on
this technique, the inventors have conducted the following
experiments in order to obtain both alleles-disrupted clones from
the TT2F antibody heavy-chain homologous recombinants #131 and
#141. First, in order to determine the lethal concentration of G418
for both #131 and #141 clones, each clone was inoculated into ten
35 mm plates at a rate of about 100 cells per plate (in this
Example, G418 resistant primary culture cells which were not
treated with mitomycin were used as feeder cells)(see Example 9).
The cells were cultured in an ES medium containing 0, 0.5, 1, 2, 3,
5, 8, 10, 15 and 20 mg/ml of G418 (GENETICIN, Sigma) for 10 days.
As a result, definite colonies were observed at a concentration of
up to 3 mg/ml, but no colony formation was observed at 5 mg/ml.
Based on these results, the minimum lethal concentration was
decided to be 5 mg/ml. Then, high concentration G418 resistant
clones were selected at concentrations of 4, 5, 6, 7 and 8 mg/ml.
For each of #131 and #141, cells were inoculated into ten 100 mm
plates at a rate of about 10.sup.6 cells per plate and cultured in
an ES medium containing G418 at each of the concentrations
described above (5 grades; two plates for each concentration).
Twelve days after the start of culture, definite colonies (#131: 12
clones; #141: 10 clones) were picked up from plates of 7 mg/ml and
8 mg/ml in G418 concentration. These clones were stored frozen and
genomic DNA was prepared by the same procedures as in Example 49.
The genomic DNAs from these high concentration G418 resistant
clones were digested with restriction enzymes EcoRI and XhoI
(Takara Shuzo) and separated by agarose gel electrophoresis. Then,
Southern blotting was performed to detect with the probe from
Section 3, Example 48 those clones in which both alleles have been
disrupted. As a result, one clone derived from #131 (#131-3) was
found to be both alleles-distrupted clone. The results of Southern
blot analysis of 6 clones derived from #131 are shown in FIG. 28.
In wild-type TT2F cells, two wild-type bands (a, b) are detected
after the EcoRI and XhoI digestion. In one allele homologous
recombinants (#131, #141), the upper band (a) has disappeared and a
new band (c) has appeared (Example 49). Furthermore, it is expected
that due to the disruption of both alleles, another wild-type band
(b) disappears and that the disruption-type band (c) remains alone.
In FIG. 28, this band pattern is observed in clone No. 3 (#131-3).
This demonstrates that both alleles of an antibody heavy-chain gene
have been disrupted in this clone.
EXAMPLE 52
[0470] Removal of a G418 Resistance Marker Gene from the Antibody
Heavy-Chain-Deficient Homozygote TT2F Clone
[0471] The G418 resistance marker gene in the antibody heavy-chain
both alleles-disrupted clone (high concentration G418 resistant
clone #131-3) from Example 51 was removed by the following
procedures. An expression vector, pBS185 (BRL), containing Cre
recombinase gene which causes a site-specific recombination between
the two LoxP sequences inserted at both the ends of the G418
resistance gene was transferred into #131-3 clone according to the
methods described in Shinichi Aizawa, "Biomanual Series 8, Gene
Targeting", published by Yodosha, 1995 and Seiji Takatsu et al.,
"Experimental Medicine (extra number): Basic Technologies in
Immunological Researches", p. 255-, published by Yodosha, 1995).
Briefly, #131-3 cells were treated with trypsin and suspended in
HBS to give a concentration of 2.5.times.10.sup.7 cells/ml. To the
cell suspension, 30 .mu.g of pBS185 DNA was added. Then,
electroporation was performed with a gene pulser (Bio-Rad
Laboratories, Inc.; resistor unit not connected). A voltage of 250
V was applied at a capacitance of 960 .mu.F using an
electroporation cell of 4 mm in length (see Example 1). The
electroporated cells were suspended in 5 ml of an ES medium and
inoculated into a tissue culture plastic plate (Corning) of 60 mm
in which feeder cells were seeded preliminarily. After two days,
the cells were treated with trypsin and reinoculated into three 100
mm plates (preliminarily seeded with feeder cells) such that the
three plates have 100, 200 and 300 cells, respectively. A similar
experiment was also conducted under the same conditions except that
the setting of the gene pulser was changed (resistor unit
connected; resistance value infinite). After seven days, a total of
96 colonies formed were picked up and treated with trypsin. Then,
the colonies were divided into two groups; one was inoculated into
a 48-well plate preliminarily seeded with feeder cells and the
other was inoculated into a 48-well plate coated with gelatin
alone. The latter was cultured in a medium containing 300 .mu.g/ml
of G418 (GENETICIN, Sigma) for three days. Then, G418 resistance
was judged from the survival ratio. As a result, 6 clones died in
the presence of G418. These G418 sensitive clones were grown to
confluence in 35 mm plates, and four fifths of the resultant
culture was suspended in 0.5 ml of a preservation medium [ES
medium+10% DMSO (Sigma)] and stored frozen at -80.degree. C. The
remaining one fifth was inoculated into a 12-well gelatin coated
plate and cultured for two days. Thereafter, genomic DNA was
prepared by the same procedures as in Example 2. These genomic DNAs
from G418 sensitive TT2F clones were digested with restriction
enzyme EcoRI (Takara Shuzo) and separated by agarose gel
electrophoresis. Then, Southern blotting was performed to confirm
the removal of the G418 resistance gene using a 3.2 kb XhoI
fragment (probe A) from G418 resistance gene-containing pSTneoB. As
a result, bands observed in #131-3 clone which hybridize with Probe
A were not detected at all in the sensitive clones. From these
results, it was confirmed that the G418 resistance marker gene had
been surely removed in the G418 sensitive clones obtained.
Additionally, as a result of Southern blot analysis performed in
the same manner using Probe B obtained by digesting pBS185 DNA with
EcoRI, no specific band which hybridizes with Probe B was detected
in these G418 sensitive clones. Thus, it is believed that Cre
recombinase-containing pBS185 is not inserted into the chromosomes
of the sensitive clones. In other words, these sensitive clones can
be transformed with the vector for knocking out an antibody
light-chain (vector having a loxP sequence at both the ends of a
G418 resistance gene) described in Section 4, Example 48.
EXAMPLE 53
[0472] Transfer of Human Chromosome #14 (Containing Antibody
Heavy-Chain Gene) into the Antibody Heavy-Chain-Deficient ES Cell
Clone
[0473] Human chromosome #14 (containing an antibody heavy-chain
gene) marked with a G418 resistance gene is transferred by
microcell fusion as described in Example 9 into the mouse ES cell
clone (from TT2F, G418 sensitive) obtained in Example 52 which is
deficient in an endogenous antibody heavy-chain. In the resultant
G418 resistant clone, the retention of human chromosome #14
(fragment) containing a human antibody heavy-chain gene is
confirmed by PCR analysis or the like (see Example 9).
EXAMPLE 54
[0474] Transfer of Human Chromosome #2 Fragment or Human Chromosome
#22 Into the Antibody Heavy-Chain-Deficient ES Cell Clone Retaining
Human Chromosome #14 (Fragment)
[0475] A human chromosome #2 fragment (containing the antibody
heavy-chain .kappa. gene) or human chromosome #22 (containing the
antibody heavy-chain .lambda. gene) marked with a puromycin
resistance gene is transferred into the antibody
heavy-chain-deficient mouse ES cell clone retaining a human
chromosome #14 partial fragment (G418 resistant) from Example 53 by
microcell fusion as described in Examples 18 and 35. In the
resultant puromycin and G418 double drug-resistant clone, the
retention of the human chromosome #14 (fragment) and human
chromosome #2 fragment or #22 (fragment) is confirmed by PCR
analysis or the like (see Examples 18 and 35).
EXAMPLE 55
[0476] Production of Chimeric Mice from the Endogenous Antibody
Heavy-Chain-Deficient Mouse ES Cells Retaining Human Chromosome #14
(Fragment) Containing a Human Antibody Heavy-Chain Gene
[0477] Chimeric mice from the endogenous antibody heavy-chain
gene-deficient mouse ES cell clone obtained in Example 53 retaining
human chromosome #14 (fragment) containing a human antibody
heavy-chain gene are produced by the same procedures as in Example
10. In the resultant chimeric mice, a human antibody heavy-chain
produced in the ES cell clone-derived B cells is detected by the
method described in Example 14. Since antibody heavy-chain genes
functional in the ES cell clone-derived B cells are only the
human-derived gene on the transferred chromosome, many of the ES
cell clone-derived B cells produce human antibody heavy-chain.
EXAMPLE 56
[0478] Production of Chimeric Mice from the Endogenous Antibody
Heavy-Chain-Deficient Mouse ES Cells Retaining Human Chromosomes
#14+#2 (Fragments) or #14+#22 (Fragments)
[0479] Chimeric mice are produced by the same procedures as in
Examples 19, 36, etc from the endogenous antibody heavy-chain
gene-deficient mouse ES cell clone retaining human chromosomes
#14+#2 (fragments) or #14+#22 (fragments) obtained in Example 54.
In the resultant chimeric mice, human antibody heavy-chain and
light-chain .kappa. or .gamma. are detected in the ES cell
clone-derived B cells according to the method described in Examples
14, 23 and 32. As in Example 55, antibody heavy-chain genes
functional in the ES cell clone-derived B cells are only the
human-derived gene on the transferred chromosome. Thus, many of the
ES cell clone-derived B cells produce human heavy-chains.
Furthermore, complete human antibody molecules both heavy and
light-chains which are derived from humans are also detected by the
method described in Examples 37 and 38.
EXAMPLE 57
[0480] Production of Human Antibody-Producing Hybridomas from the
Chimeric Mice Derived from the Endogenous Antibody
Heavy-Chain-Deficient Mouse ES Cells Retaining Human Chromosomes
#14+#2 (Fragments) or #14+#22 (Fragments)
[0481] The chimeric mice from Example 56 are immunized with an
antigen of interest in the same manner as in Examples 15, 25 and
34. The spleen is isolated from each mice and the spleen cells are
fused with myeloma cells to produce hybridomas. After cultivation
for 1-3 weeks, the culture supernatant is analyzed by ELISA. The
ELISA is performed by the method described in Examples 14, 15, 21,
24, 25, 33, 34, 37 and 38. As a result, human antibody positive
clones and clones which are human antibody positive and specific to
the antigen used in the immunization are obtained.
EXAMPLE 58
[0482] Production of an Antibody Light-Chain Gene-Disrupted Clone
from the Antibody Heavy-Chain-Deficient Homozygote Mouse ES
Cells
[0483] A homologous recombinant, which has further disruption in an
antibody light-chain gene in the antibody heavy-chain-deficient
homozygote TT2F cell clone (G418 sensitive) obtained in Example 52
is produced by the following procedures. Briefly, the antibody
light-chain targeting vector prepared in Section 4, Example 48 is
linearized with restriction enzyme KpnI (Takara Shuzo), and
transferred into the above TT2F cell clone (G418 sensitive)
according to the method described in Shinichi Aizawa, "Biomanual
Series 8: Gene Targeting", published by Yodosha, 1995. After 7-9
days, colonies formed are picked up. They are stored frozen and
genomic DNA is prepared in the same manner as in Example 49.
Genomic DNAs from G418 resistant clones are digested with
restriction enzymes EcoRI and NotI (Takara Shuzo) and separated by
agarose gel electrophoresis. Then, Southern blot analysis is
performed to detect homologous recombinants with the probe
described in Section 4, Example 48.
EXAMPLE 59
[0484] Production of an Double Knockout Clone from the Antibody
Light-Chain Homologous Recombinant
[0485] A clone in which both alleles of a light-chain gene are
disrupted is prepared from the TT2F antibody light-chain homologous
recombinant (and antibody heavy-chain-deficient homozygote) clone
from Example 58 by the procedures described below. Briefly, a high
concentration G418 resistant clone is prepared and stored frozen,
and DNA is prepared in the same manner as in Example 51. Genomic
DNA from the high concentration G418 resistant clone is digested
with restriction enzymes EcORI and NotI (Takara Shuzo) and
separated by agarose gel electrophoresis. Then, Southern blot
analysis is performed to detect those clones in which both alleles
have been disrupted, with the probe from Section 4, Example 48.
EXAMPLE 60
[0486] Removal of the G418 Resistance Gene from the Antibody
Light-Chain-Deficient Homozygote (Antibody Heavy-Chain-Deficient
Homozygote) TT2F Cell Clone
[0487] The G418 resistance marker gene in the antibody light-chain
both alleles-disrupted clone (high concentration G418 resistant
clone) obtained in Example 59 is removed by the same procedures as
in Example 52. Briefly, an expression vector, pBS185 (BRL),
containing Cre recombinase gene which causes a site-specific
recombination between the two loxp sequences inserted at both the
ends of the G418 resistance gene (Section 1, Example 48) was
transferred into the above clone according to the method described
in Example 52. The resultant G418 sensitive clones are grown to
confluence in 35 mm plates, and 4/5 of the resultant culture was
suspended in 0.5 ml of a preservation medium [ES medium+10% DMSO
(Sigma)] and stored frozen at -80.degree. C. by the same procedures
as in Example 52. The remaining 1/5 was inoculated into a 12-well
gelatin coated plate. After cultivation for two days, genomic DNA
is prepared by the method described in Example 2. These genomic
DNAs from G418 sensitive TT2F clones are digested with restriction
enzyme EcoRI (Takara Shuzo) and separated by agarose gel
electrophoresis. Then, Southern blotting is performed to confirm
the removal of the G418 resistance gene using a 3.2 kb XhoI
fragment from G418 resistance gene-containing pSTneoB as a
probe.
EXAMPLE 61
[0488] (1) Transfer of a Human Chromosome #14 Fragment (Containing
Antibody Heavy-Chain Gene) into the Endogenous Antibody Heavy-Chain
and .kappa. Chain-Deficient ES Cell Clone
[0489] A human chromosome #14 fragment SC20 (containing a human
antibody heavy-chain gene) was transferred by microcell fusion as
described in Section 2 of Example 68 into the mouse ES cell clone
HKD31 (from TT2F, G418 sensitive, puromycin sensitive) obtained in
Example 78 which is deficient in both endogenous antibody
heavy-chain and X chain. The microcell fusion and the selection of
G418 resistant clones were performed in the same manner as in
Example 2. Eight of the resultant G418 resistant clones were
subjected to PCR analysis using IgM and D14S543 primers (see
Example 68). As a result, both markers were detected in 8 out of
the 7 clones analyzed. Hence, it was confirmed that the antibody
heavy-chain and .kappa. chain-deficient ES cell clone retains the
human chromosome #14 fragment SC20.
[0490] (2) Production of Chimeric Mice from the Endogenous Antibody
Heavy-Chain and .kappa. Chain Genes-Disrupted Mouse ES Cells
Retaining a Human Chromosome #14 Fragment (Containing Antibody
Heavy-Chain Gene)
[0491] Chimeric mice were produced by the same procedures as in
Example 10, etc. from the endogenous antibody heavy-chain and
.kappa. chain genes-disrupted mouse ES cell clone HKD31-8 which was
obtained in Section 1 of Example 61 and which retains a human
chromosome #14 fragment (containing a human antibody heavy-chain
gene). As a result of transplantation of a total of 188 injected
embryos, 25 offspring mice were born. Chimerism in the offsprings
can be determined by the extent of TT2 cell-derived agouti coat
color (dark brown) in the host embryo (ICR)-derived albino coat
color (white). Out of the 25 offsprings, 17 mice were recognized to
have partial agouti coat color, indicating the contribution of the
ES cells. Out of the 17 mice, three were chimeric mice in which
more than 95% of their coat color was (ES cell-derived) agouti.
[0492] From these results, it was confirmed that the endogenous
antibody heavy-chain and .kappa. chain genes-disrupted mouse ES
cell clone retaining the human chromosome #14 fragment (containing
a human antibody heavy-chain gene) maintains the ability to produce
chimera, that is, the ability to differentiate into normal tissues
of mice.
[0493] (3) Detection of Human Antibody (Having Human .mu., .gamma.
or .alpha. Chain) in sera of the Chimeric Mice Derived from the
Endogenous Antibody Heavy-Chain and .kappa. Chain Genes-Disrupted
Mouse ES Cells Retaining a Human Chromosome #14 Fragment
(Containing Antibody Heavy-Chain Gene)
[0494] The chimeric mice produced in Section 2 of Example 61
(derived from HKD31-8) were bled 12 weeks (#1) or 7 weeks (#2-4)
after birth. The human antibody concentration in the sera was
determined by ELISA in the same manner as in Example 14. Ninety
six-well microtiter plates were coated with PBS-diluted anti-human
immunoglobulin .mu. chain antibody (Sigma, I6385) or anti-human
immunoglobulin .gamma. chain antibody (Sigma, I3382) or anti-human
immunoglobulin .alpha. chain antibody (Pharmingen, 08091D) and then
a serum sample diluted with mouse serum (Sigma, M5905)-containing
PBS was added. Subsequently, peroxidase-labeled anti-human
immunoglobulin .mu. chain antibody (The Binding Site Limited,
MP008) or peroxidase-labeled anti-human immunoglobulin .gamma.
chain antibody (Sigma, A0170) was added to the plates and
incubated. Alternatively, biotin-labeled anti-human immunoglobulin
.alpha. chain antibody (Pharmingen, 08092D) was added to the plates
and incubated. After the plates were washed, an avidin-peroxidase
complex (Vector, ABC Kit PK4000) was added thereto and incubated.
TMBZ (Sumitomo Bakelite, ML-1120T) was added as a peroxidase
substrate and then enzyme activity was determined by absorbance
measurement at 450 nm. Purified human immunoglobulins IgM (CAPPEL,
6001-1590), IgG (Sigma, I4506) and IgA (Sigma, I2636) of known
concentrations having .mu. chain, .gamma. chain and .alpha. chain,
respectively, were used as standards for determining human antibody
concentrations in the sera. These standards were diluted stepwise
with mouse serum-supplemented PBS. The results are shown in Table
20. Chimeric mice having concentrations of human antibody .mu. and
.lambda. chains almost as high as in normal mouse sera were
confirmed. Also, chimeric mice expressing human .alpha. chain were
confirmed. Further, human immunoglobulin .gamma. chain sub-classes
were detected in the same manner as in Example 29. As a result, all
of the four subclasses (.gamma.1, .gamma.2, .gamma.3 and .gamma.4)
were detected.
[0495] These results show that a human antibody heavy-chain gene is
expressed efficiently in the chimeric mice derived from the
endogenous antibody heavy-chain and .kappa. chain genes-disrupted
mouse ES cells retaining the human chromosome #14 fragment
(containing an antibody heavy-chain gene); it was also shown that
not only .mu. chain but also all of the .gamma. chain subclasses
and .alpha. chain were expressed therein as a result of class
switching.
29TABLE 20 Human Antibody Concentrations in Chimeric Mice (ELISA)
Chimeric Chimerism Human Antibody (mg/l) Mouse % IgM IgG IgA #1 90
270 1250 0.46 #2 99 370 820 0.23 #3 99 550 1460 0.32 #4 95 340 2300
0.06
[0496] (4) Acquisition of Hybridomas Producing Anti-HSA Antibody
Comprising Human .gamma. Chain from the Chimeric Mice Derived from
the Endogenous Antibody Heavy-Chain and .kappa. Chain
Genes-Disrupted Mouse ES Cells Retaining a Human Chromosome #14
Fragment (Containing Antibody Heavy-Chain Gene)
[0497] Chimeric mice #3 (derived from HKD31-8; chimerism 99%) and
#4 (chimerism 95%) which had exhibited a high human antibody
.gamma. chain concentration in the serum in Section 3 of Example 61
were immunized as described below. Human serum albumin (HSA, Sigma,
A3782) dissolved in PBS was mixed with an adjuvant (MPL+TDM
Emulsion, RIBI Immunochem Research Inc.) to prepare an HSA solution
with a concentration of 0.25 mg/ml. When the above-described
chimeric mice became 16-week old, 0.2 ml of this HSA solution was
administered intraperitoneally twice at an interval of 2 weeks. Two
weeks thereafter, the mice were immunized with human serum albumin
dissolved in PBS and then bled. The concentration of anti-HSA human
antibody in the sera was determined by ELISA in the same manner as
in Example 14. Briefly, ELISA plates were coated with HSA and then
peroxidase-labeled anti-human Ig.mu. antibody (The Binding Site,
MP008), anti-human Ig.gamma. antibody (Sigma, A1070) and anti-human
Ig.alpha. antibody (Kirkegaard & Perry Laboratories Inc.,
14-10-01) were used for detection. The results are shown FIG. 33.
Hybridomas were produced using a myeloma cell SP-2/0-Ag14
(Dainippon Pharmaceutical Co., Ltd.) by the method described in
Ando, "Monoclonal Antibody Experiment Procedure Manual", published
by Kodansha Scientific in 1991. Three days after the final
immunization, the spleen was removed from the chimeric mice and
then cell fusion was performed using PEG in the same manner as in
Example 29 to prepare hybridomas. At the same time, blood samples
were collected from the mice to quantitate human Ig.gamma.
subclasses in the sera. As a result, 920 mg/l of .gamma. 1, 520
mg/l of .gamma. 2, 11 mg/l of .gamma. 3 and 140 mg/l of .gamma. 4
were detected in the serum of chimeric mouse #3.
[0498] The fused cells were diluted with a medium (Sanko Pure
Chemical, S Cloning Medium CM-B) containing 5% HCF (Air Brown) and
HAT (Dainippon Pharmaceutical Co., Ltd., No. 16-808-49) or 1 mg/ml
of G418 to give a concentration of 10.sup.6 spleen cells/ml and
then dispensed into 96-well plates (100 .mu.l/well), followed by
cultivation. At day 8 of the cultivation, the culture supernatant
was collected and screened for human antibody-producing hybridomas
by ELISA in the same manner as in Example 14. Briefly, ELISA plates
were coated with a HSA solution dissolved in CBB buffer to give a
concentration of 5.mu.g/ml. Peroxidase-labeled anti-human
immunoglobulin .gamma. chain antibody (Sigma, A0170) and TMBZ
(Sumitomo Bakelite, ML-1120T) were used for detection. An
absorbance about 3 times higher than the absorbance in the negative
control was used as a criterion for judgement. As a result, 74
positive wells were obtained from chimeric mouse #3 and 29 positive
wells from chimeric mouse #4. Also, anti-HSA antibody having human
.mu. chain was screened in HSA-solution-coated plates using
peroxidase-labeled anti-human immunoglobulin .mu. chain antibody
(Tago, #2392). Briefly, fused cells from chimeric mouse #3 were
inoculated into fifteen 96-well plates, from which 4 plates were
selected by G418 resistance. The culture supernatants of these 4
plates were screened to obtain 5 positive wells. Wells which
exhibited colony formation after selection with HAT or 1 mg/ml of
G418 were 74 wells/plate for HAT and 29 wells/plate for G418. The
cells of those wells which were positive for human .gamma.
chain-containing anti-HSA antibody and which had a relatively large
number of cells were transferred into 46-well plates and cultured
for another 4 days. The isotype of the antibody in the culture
supernatant was determined by ELISA. ELISA was performed in
HSA-coated plates using alkali phosphatase-labeled anti-human IgG1
antibody (Zymed Labolatories, Inc., 05-3322), anti-human IgG2
antibody (Zymed Labolatories, Inc., 05-3522), anti-human IgG3
antibody (Zymed Labolatories, Inc., 05-3622) and anti-human IgG4
antibody (Zymed Labolatories, Inc., 05-3822) in the same manner as
in Example 14. As a result, 27 human IgG1 positive clones, 11 human
IgG2 positive clones, 2 human IgG3 positive clones and 13 human
IgG4 positive clones were obtained. Fused cells from chimeric mouse
#4 were treated in the same manner to obtain 4 positive clones with
a large number of cells as human IgG1 producing clones.
[0499] These results show that the immunization by human protein
(HSA) of the chimeric mice derived from the endogenous antibody
heavy-chain & light-chain-deficient mouse ES cells retaining
the human chromosome #14 partial fragment containing a human
antibody heavy-chain gene increases the antibody titers of antigen
specific human Ig.mu., .gamma. and .alpha. to thereby enable the
acquisition of hybridomas producing anti-HSA antibody containing
.mu. chain and all of the human .gamma. chain subclasses.
EXAMPLE 62
[0500] Transfer of Human Chromosome #2 (Containing Light-Chain
.kappa. Gene) Into the Endogenous Antibody Heavy-Chain and .kappa.
Chain-Deficient ES Cells Retaining a Human Chromosome #14 Fragment
(Containing Antibody Heavy-Chain Gene)
[0501] A human chromosome #2 fragment (containing antibody
light-chain .kappa. gene) marked with a puromycin resistance gene
was transferred into the endogenous antibody heavy-chain and
.kappa. chain-deficient mouse ES cell clone HKD31-8 obtained in
Section 1 of Example 61 and which retained a human chromosome #14
fragment (containing an antibody heavy-chain gene). The method of
transfer was by microcell fusion as described in Example 18. As a
result, 13 puromycin and G418 double-resistant clones were
obtained. These clones were subjected to PCR analysis (see Example
18) using IgM and D14S543 primers (see Example 68) for the
chromosome #14 fragment and V.kappa. 1 and FABP1 primers (see
Example 12) for the chromosome #2 fragment. As a result, the
presence of all the 4 markers was confirmed in 8 clones. Of these
clones, KH13 clone was subjected to FISH analysis using human
chromosome-specific probes (see Examples 9 and 12). The results are
shown in FIG. 34. Two independent, small chromosome fragments
hybridizing to the probes were observed in KH13. These results show
that KH13 retains both the chromosome #14 fragment and the
chromosome #2 fragment.
EXAMPLE 63
[0502] Transfer of Human Chromosome #22 (Containing Light-Chain
.lambda. Gene) Into the Endogenous Antibody Heavy-Chain and .kappa.
Chain-Deficient ES Cells Retaining a Human Chromosome #14 Fragment
(Containing Antibody Heavy-Chain Gene)
[0503] Human chromosome #22 (containing antibody light-chain
.lambda. gene) marked with a puromycin resistance gene was
transferred into mouse ES cell clone HKD31-8 obtained in Section 1
of Example 61 which was deficient in the endogenous antibody
heavy-chain & .kappa. chain and which retained a human
chromosome #14 fragment (containing an antibody heavy-chain gene).
The method of transfer was by microcell fusion as described in
Example 35. As a result, 12 puromycin and G418 double
drug-resistant clones were obtained. These clones were subjected to
PCR analysis (see Example 35) using IgM and D14S543 primers for the
chromosome #14 fragment and Ig .lambda., D22S315, D22S275, D22S278,
D22S272 and D22S274 primers (see Example 2) for the chromosome #22
fragment. As a result, the presence of all of the 8 markers was
confirmed in 10 clones. Of the remaining 2 clones, LH13 clone
exhibited the presence of 5 markers, IgM, D14S543, IgI, D22S275 and
D22S274. Thus, it is believed that this clone contains a fragment
of human chromosome #22. LH13 was further subjected to FISH
analysis using a human chromosome #22-specific probe and a human
chromosome #14-specific probe separately. As a result, independent
chromosome fragments hybridizing to the respective probes were
observed. This indicates that this clone retains both a chromosome
#14 fragment and a chromosome #2 fragment.
EXAMPLE 64
[0504] Production of Endogenous Antibody Heavy-Chain &
Light-Chain-Deficient Mouse ES Cells Retaining Three Human
Chromosomes, #2 (Containing Antibody Light-Chain .kappa. Gene), #14
(Containing Antibody Heavy-Chain Gene) and #22 (Containing Antibody
.lambda. Chain Gene), or Partial Fragments Thereof
[0505] In order to obtain mouse ES cells retaining three kinds of
human chromosomes, human chromosome #2 or #22 is marked by
inserting a marker gene such as blasticidin resistance (Izumi et
al., Exp. Cell. Res., 197: 229, 1991), hygromycin resistance (Wind
et al., Cell, 82:321-, 1995), etc. This marking is performed
according to the method described in Examples 16 and 26. Human
chromosome #22 (containing human antibody light-chain .lambda.
gene) marked with blasticidin resistance, hygromycin resistance,
etc. is transferred into the mouse ES cell clone (from TT2F, G418
resistant, puromycin resistant) obtained in Example 62 which is
deficient in endogenous antibody heavy-chain & light-chain and
which retains both human chromosome #14 (fragment) and human
chromosome #2 (partial fragment). The method of transfer is by the
method described in Example 9. As feeder cells for culturing ES
cells, appropriate cells are selected depending on the selection
marker used. When a hygromycin resistance marker is used, primary
culture fibroblasts obtained from a transgenic mouse strain which
retains and expresses the marker (Johnson et al., Nucleic Acids
Research, vol. 23, No. 7, 1273-, 1995) are used. It is confirmed by
PCR analysis, etc. (see Examples 9, 18 and 35) that the resultant
G418, puromycin and hygromycin (or blasticidin) triple
drug-resistant clones retain the three kinds of human chromosomes
(fragments) described above. In the same manner, a human chromosome
#2 fragment marked with a hygromycin or blasticidin resistance gene
is transferred into the mouse ES cell clone (from TT2F, G418
resistant, puromycin resistant) obtained in Example 63 which is
deficient in endogenous antibody heavy-chain & light-chain and
which retains both human chromosome #14 (fragment) and human
chromosome #22 (fragment).
EXAMPLE 65
[0506] Production of Chimeric Mice from the Endogenous Antibody
Heavy-Chain & Light-Chain Genes-Disrupted Mouse ES Cells
Retaining a Plurality of Human Chromosomes (Fragments) Containing
Human Antibody Heavy-Chain Gene and Light-Chain Gene,
Respectively
[0507] Chimeric mice are produced by the same procedures as in
Example 10, etc. from the endogenous antibody heavy-chain &
light-chain genes-disrupted mouse ES cell clones that retain human
chromosomes (fragments) containing human antibody genes and which
were obtained in Examples 61, 62, 63 and 64. In the resultant
chimeric mice, mouse antibodies produced in host embryo-derived B
cells and human antibodies produced mainly in ES cell clone-derived
B cells are detected by the method described in Examples 14, 23 and
32. Since the antibody heavy-chain gene and the light-chain .kappa.
gene which are both functional in the ES cell clone-derived B cells
are only human-derived genes on the transferred chromosomes, many
of the ES cell clone-derived B cells produce human antibody
heavy-chain and light-chain .kappa. (Lonberg et al., Nature,
368:856-, 1994). Furthermore, complete human antibody molecules in
which both heavy- and light-chains are derived from human are also
detected by the method described in Examples 37 and 38.
[0508] (1) Production of Chimeric Mice from the Endogenous Antibody
Heavy-Chain and .kappa. Chain Genes-Disrupted Mouse ES Cells
Retaining Both a Human Chromosome #14 Fragment (Containing Antibody
Heavy-Chain Gene) and a Human Chromosome #2 Fragment (Containing
Antibody Light-Chain .kappa. Gene)
[0509] Chimeric mice were produced by the same procedures as in
Example 10, etc. from mouse ES cell clone KH13 obtained in Example
62 which is deficient in the endogenous antibody heavy-chain and
.kappa. chain genes and which retains both a human chromosome #14
fragment (containing an antibody heavy-chain gene) and a human
chromosome #2 fragment (containing light-chain .kappa. gene). As a
result of transplantation of a total of 176 injected embryos, 20
offspring mice were born. Chimerism in the offsprings can be
determined by the extent of TT2 cell-derived agouti coat color
(dark brown) in the host embryo (ICR)-derived albino coat color
(white). Out of the 20 offsprings, 7 mice were recognized to have
partial agouti coat color, indicating the contribution of the ES
cells.
[0510] From-these results, it was confirmed that the endogenous
antibody heavy-chain and .kappa. chain genes-disrupted mouse ES
cell clone retaining both a human chromosome #14 fragment
(containing an antibody heavy-chain gene) and a human chromosome #2
fragment (containing light-chain .kappa. gene) maintains the
ability to produce chimera, that is, the ability to differentiate
into normal tissues of mice.
[0511] (2) Production of Chimeric Mice from the Endogenous Antibody
Heavy-Chain and .kappa. Chain Genes-Disrupted Mouse ES Cells
Retaining Both a Human Chromosome #14 Fragment (Containing Antibody
Heave-Chain Gene) and a Human Chromosome #22 Fragment (Containing
Light-Chain .lambda. Gene)
[0512] Chimeric mice were produced by the same procedures as in
Example 10, etc. from mouse ES cell clone LH13 obtained in Example
63 which is deficient in the endogenous antibody heavy-chain and
.kappa. chain genes and which retains both a human chromosome #14
fragment (containing an antibody heavy-chain gene) and a human
chromosome #22 fragment (containing light-chain .lambda. gene). As
a result of transplantation of a total of 114 injected embryos, 22
offspring mice were born. Chimerism in the offsprings can be
determined by the extent of TT2 cell-derived agouti coat color
(dark brown) in the host embryo (ICR)-derived albino coat color
(white). Out of the 22 offsprings, 5 mice were recognized to have
partial agouti coat color, indicating the contribution of the ES
cells.
[0513] From these results, it was confirmed that the endogenous
antibody heavy-chain and .kappa. chain genes-disrupted mouse ES
cell clone retaining both a human chromosome #14 fragment
(containing an antibody heavy-chain gene) and a human chromosome
#22 fragment (containing light-chain .lambda. gene) maintains the
ability to produce chimera, that is, the ability to differentiate
into normal tissues of mice.
[0514] (3) Detection and Quantitative Determination of Complete
Human Antibody in sera of the Chimeric Mice Derived from the
Endogenous Antibody Heavy-Chain and .kappa. Chain-Deficient Mouse
ES Cells Retaining Both a Human Chromosome #2 Partial Fragment and
a Human Chromosome #14 Partial Fragment
[0515] The chimeric mice (derived from KH13) produced in Section 1
of Example 65 were bled at day 40 after birth. The concentrations
of human antibody in the sera were determined by ELISA in the same
manner as in Example 14. Briefly, ELISA plates were coated with
PBS-diluted anti-human immunoglobulin .kappa. chain antibody
(Kirkegaard & Perry Labolatories Inc., 01-10-10) or anti-human
immunoglobulin .kappa. chain antibody (Vector, AI-3060) and then
serum samples diluted with mouse serum (Sigma, M5905)-supplemented
PBS were added. Subsequently, peroxidase-labeled anti-human
immunoglobulin g chain antibody (The Binding Site Limited, MP008)
or peroxidase-labeled anti-human immunoglobulin .gamma. chain
antibody (Sigma, A0170) was added and incubated. TMBZ (Sumitomo
Bakelite, ML-1120T) was added as a peroxidase substrate and then
enzyme activity was determined by absorbance measurement at 450 nm.
Purified human immunoglobulins IgM (Caltag, 13000) and IgG (Sigma,
I4506) of known concentrations having .mu. chain and .kappa. chain
were used as standards for determining human antibody
concentrations in the sera by comparison. These standards were
diluted stepwise with mouse serum-supplemented PBS. The results are
shown in Table 21. Chimeric mice were confirmed that had
concentrations of complete human antibody more than 10 times higher
than in chimeric mice derived from ES cells whose endogenous
antibody genes were not knocked out. Also, complete human antibody
containing human.sub..gamma. chain was confirmed in the sera of the
chimeric mice.
[0516] From these results, it was confirmed that the concentration
of complete human antibody in which both heavy- and light-chains
were derived from human increasesed in the chimeric mice derived
from the endogenous antibody heavy-chain and K chain-deficient
mouse ES cells retaining both a human chromosome #14 partial
fragment and a human chromosome #22 partial fragment.
30TABLE 21 Concentrations of Human Antibodies in Chimeric Mice
(ELISA) ES Chimeric clone mouse Chimerism (%) IgM, .kappa. (mg/l)
IgG, .kappa. (mg/l) KH13 CKH13-1 95 0.1 0.07 KH13 CKH13-2 85 0.9
0.13
[0517] (4) Detection and Quantitative Determination of Complete
Human Antibody in sera of the Chimeric Mice Derived from the
Endogenous Antibody Heavy-Chain and .kappa. Chain-Deficient Mouse
ES Cells Retaining Both a Human Chromosome #14 Partial Fragment and
a Human Chromosome #22 Partial Fragment
[0518] The chimeric mice (derived from KH13) produced in Section 2
of Example 65 were bled at day 49 after birth. The concentrations
of human antibody in the sera were determined by ELISA in the same
manner as in Example 14. Briefly, ELISA plates were coated with
PBS-diluted anti-human immunoglobulin .lambda. chain antibody
(Kirkegaard & Perry Labolatories Inc., 01-10-11) or anti-human
immunoglobulin .lambda. chain antibody (Vector, AI-3070) and then
serum samples diluted with mouse serum (Sigma, M5905)-supplemented
PBS were added. Subsequently, peroxidase-labeled anti-human
immunoglobulin .mu. chain antibody (The Binding Site Limited,
MP008) or peroxidase-labeled anti-human immunoglobulin .gamma.
chain antibody (Sigma, A0170) was added and incubated. TMBZ
(Sumitomo Bakelite, ML-1120T) was added as a peroxidase substrate
and then enzyme activity was determined by absorbance measurement
at 450 nm. Purified human immunoglobulins IgM (Caltag, 13000) and
IgG (Sigma, I4506) of known concentrations having .mu. chain and
.kappa. chain were used as standards for determining human antibody
concentrations in the sera by comparison. These standards were
diluted stepwise with mouse serum-supplemented PBS. The results are
shown in Table 22. Chimeric mice individuals were confirmed that
had concentrations of complete human antibody about 40 times higher
than in chimeric mice derived from ES cells whose endogenous
antibody genes were not knocked out. Also, complete human antibody
containing human .gamma. chain was confirmed in the sera of the
chimeric mice.
[0519] From these results, it was confirmed that the concentration
of complete human antibody in which both heavy- and light-chains
were derived from human increasesed in the chimeric mice derived
from the endogenous antibody heavy-chain and .kappa.
chain-deficient mouse ES cells retaining both a human chromosome
#14 partial fragment and a human chromosome #22 partial
fragment.
31TABLE 22 Concentrations of Human Antibodies in Chimeric Mice
(ELISA) ES Chimeric clone mouse Chimerism (%) IgM, .lambda. (mg/l)
IgG, .lambda. (mg/l) LH13 CLH13-1 95 13 2.6 LH13 CLH13-2 90 2.8
0.36
EXAMPLE 66
[0520] Production of Complete Human Antibody-Producing Hybridomas
from Chimeric Mice Prepared by Transferring the Endogenous Antibody
Heavy-Chain and Light-Chain-Deficient Mouse ES Cells Retaining Both
a Human Chromosome #14 Partial Fragment and a Human Chromosome #22
Partial Fragment Into Immunodeficient Mouse Host Embryos
[0521] A chimeric mouse CLH13-3 (derived from TT2FES clone LH13;
chimerism 35%) obtained in Section 3 of Example 67 was immunized
with HSA from day 43 after birth. Briefly, human serum albumin
(HSA, Sigma, A3782) dissolved in PBS was mixed with an adjuvant
(MPL+TDM Emulsion, RIBI Immunochem Research Inc.) to prepare a HSA
solution with a concentration of 0.25 mg/ml, 0.2 ml of which was
administered intraperitoneally twice at an interval of 1 week. One
week thereafter, the mouse was immunized with human serum albumin
dissolved in PBS. The mouse was bled every 1 week to determine the
concentrations of anti-HSA human antibodies in the serum by ELISA
in the same manner as in Example 14. The results are shown in FIG.
35. The spleen was removed from the chimeric mouse at day 3 after
the final immunization and then cell fusion was performed using PEG
in the same manner as in Example 24 to prepare hybridomas. Briefly,
the fused cells were diluted with a medium (Sanko Pure Chemical, S
Cloning Medium CM-B) containing HAT (Dainippon Pharmaceutical Co.,
Ltd., No. 16-808-49) or 1 mg/ml of G418 to give a concentration of
10.sup.6 spleen cells/ml and then dispensed into 96-well plates
(100 .mu.l/well), followed by cultivation. Both of the selection
media contained 5% HCF (Air Brown). At day 6 of the cultivation,
colonies were formed in almost all wells in both the G418 selection
and HAT selection plates. A total of about 770 hybridoma-positive
wells were obtained. The culture supernatants were collected and
subjected to screening for human antibody-producing hybridomas by
ELISA in the same manner as in Example 14. Briefly, ELISA plates
were coated with anti-human immunoglobulin .lambda. chain antibody
(Vector, AI-3070). Biotin-labeled anti-human immunoglobulin
.lambda. chain antibody (Vector, BA-3070) and an avidin-peroxidase
complex (Vector ABC Kit PK4000) were used for detection with TMBZ
(Sumitomo Bakelite, ML-1120T) used as a substrate. An absorbance
about 2 times higher than the absorbance in the negative control
was used as a criterion for judgement. As a result, 17 positive
wells were obtained. The cells of the positive wells were
transferred into 24-well plates and cultured in IMDM medium
containing 10% FBS. The culture supernatants were analyzed by ELISA
in the same manner as in Section 4 of Example 65. As a result, the
presence of 0.09-11 mg/ml of complete human antibody having both
human Ig .mu. & Ig.lambda. was confirmed in 16 wells. The
antibody titer of anti-HSA human .lambda. chain was determined in
the same manner as in Example 33 to obtain one positive well. The
cells of the well which was complete human antibody-positive and
anti-HSA human .lambda. chain-positive were cloned by limiting
dilution according to the method described in Ando, "Monoclonal
Antibody Experiment Procedure Manual", published by Kodansha
Scientific in 1991. As a result, 2 clones of anti-HSA human
.lambda. chain-positive hybridomas were obtained.
[0522] From these results, it was confirmed that complete human
antibody-producing hybridomas could be obtained from chimeric mice
prepared by transferring the endogenous antibody heavy-chain and
light-chain-deficient mouse ES cells retaining both a human
chromosome #14 partial fragment and a human chromosome #22 partial
fragment into immunodeficient mouse host embryos. Furthermore, it
was confirmed that the antibody titers of antigen-specific human
Ig.mu. and Ig.lambda. increased in response to the stimulation with
the HSA antigen. It was further confirmed that hybridomas producing
a HAS-specific antibody consisting of human Ig.mu. and Ig.lambda.
could be obtained from this chimeric mouse.
[0523] Since the fused cells had a drug resistance marker on their
chromosome, it was possible to select hybridomas using G418 without
adding HAT. After G418 selection, only those cells having a human
chromosome grow and, thus, hybridomas can be obtained selectively.
Also, it is expected that a human chromosome can be prevented from
falling off fused cells. Furthermore, it is expected that even
myeloma cells unsuitable for HAT selection such as those having
HGPRT (hypoxanthine-guanine-phosphoribosyltransferase) enzyme may
become available for cell fusion.
EXAMPLE 67
[0524] Production of Chimeric Mice with Heavy-Chain Gene-Disrupted
Host Embryos
[0525] From those mice exhibiting agouti coat color among the
progeny of the endogenous antibody heavy-chain one allele-disrupted
TT2F cell clone-derived chimeric mice produced in Example 49, mice
retaining the disrupted allele are selected by Southern blot
analysis (Example 49) or the like (the expected possibility is
1/2). Offsprings born by the mating of those antibody
heavy-chain-deficient heterozygous male and female mice are
subjected to Southern blot analysis (see Example 49), analysis of
the production of antibody heavy-chains in sera (Kitamura et al.,
Nature, 350:423-, 1991), etc. Thus, antibody heavy-chain-deficient
homozygotes can be obtained which are deficient in both alleles and
which can hardly produce functional antibodies of their own (the
expected possibility is {fraction (1/4;)} for the results in
membrane-type .mu. chain-deficient mice, see Kitamura et al.,
Nature, 350:423-, 1991).
[0526] (1) Establishment of an Antibody Heavy-Chain Knockout Mouse
Strain
[0527] Those mice that exhibited agouti coat color among the
progeny of the endogenous antibody heavy-chain one allele-disrupted
TT2F cell clone-derived chimeric mice produced in Example 49 were
subjected to Southern blot analysis (Example 49) to select those
mice that retained the disrupted allele. Offsprings born by the
mating of these antibody heavy-chain-deficient heterozygous male
and female mice were subjected to Southern blot analysis (see
Example 49) and analysis of the production of antibody .mu. chain
in sera (Kitamura et al., Nature, 350: 423-, 1991), etc. As a
result, antibody heavy-chain-deficient homozygotes could be
obtained which were deficient in both alleles and which could
hardly produce functional antibodies of their own (for the results
in membrane-type .mu. chain-deficient mice, see Kitamura et al.,
Nature, 350:423-, 1991).
[0528] Thus, an antibody heavy-chain knockout mouse strain could be
established from the antibody heavy-chain one allele-disrupted TT2F
cell clone.
[0529] Embryos obtained by mating the homozygous male and female
mice bred in a clean environment may be used as hosts for producing
chimeric mice. In this case, most of the B cells functional in the
resultant chimeric mice are derived from the injected ES cells.
Other mouse strains which cannot produce their own functional B
cells, such as RAG-2-deficient mouse (Sinkai et al., Cell, 68:855-,
1992), may also be used for this purpose. In this system, chimeric
mice are produced by the same procedures as in Example 10, etc.
using the mouse ES cell clone from Examples 62, 63 or 64 which is
deficient in endogenous antibody heavy-chain & light-chain and
which retains human chromosomes #14+#2, #14+#22 or #14+#2+#22
(fragments). The resultant chimeric mice mainly produce human
antibodies by the expression of human antibody heavy-chain (on
chromosome #14), light-chain .kappa. (on chromosome #2) and
light-chain .lambda. (on chromosome #22) genes that are functional
in ES cell-derived B cells.
[0530] (2) Detection and Quantitative Determination of Complete
Human Antibody in sera of the Chimeric Mice Produced by Injecting
the Endogenous Antibody Heavy-Chain and .kappa. Chain-Deficient
Mouse ES Cells Retaining Both a Human Chromosome #2 Partial
Fragment and a Human Chromosome #14 Partial Fragment into
Immunodeficient Mouse Host Embryos
[0531] Chimeric mice were produced in the same manner as in Section
1 of Example 65 by injecting the ES cell clone KH10 from Example 62
into the embryos obtained by mating male and female mice of the
antibody heavy-chain knockout mouse strain established in Section 1
of Example 67. Seven-week old resultant chimeric mice were bled to
determine the concentrations of human antibodies in the sera by
ELISA in the same manner as in Example 14 and Section 3 of Example
65. The results are shown in Table 23. Complete human antibodies
having human .mu. chain+.kappa. chain and human .gamma.
chain+.kappa. chain, respectively, were confirmed in the sera of
the chimeric mice. It was also confirmed that by transferring ES
cells into immunodeficient host embryos, complete antibodies could
be obtained even in the resultant chimeric mice of low chimerism
since B cells are differentiated only from ES cells.
32TABLE 23 Concentrations of Human Antibodies in Chimeric Mice
(ELISA) Chimeric ES clone mouse Chimerism (%) IgM, .kappa. (mg/l)
IgG, .kappa. (mg/l) KH13 CKH10-1 6 6.1 0.17 KH13 CKH10-2 3 1.9
0.4
[0532] (3) Detection and Quantitative Determination of Complete
Human Antibody in sera of the Chimeric Mice Produced by Injecting
the Endogenous Antibody Heavy-Chain and .kappa. Chain-Deficient
Mouse ES Cells Retaining Both a Human Chromosome #14 Partial
Fragment and a Human Chromosome #22 Partial Fragment into
Immunodeficient Mouse Host Embryos
[0533] Chimeric mice were produced in the same manner as in Section
2 of Example 65 by injecting the ES cell clone LH13 from Example 62
into the embryos obtained by mating male and female mice of the
antibody heavy-chain knockout mouse strain established in Section 1
of Example 67. Five-week old resultant chimeric mice were bled to
determine the concentrations of human antibodies in the sera by
ELISA in the same manner as in Example 14 and Section 4 of Example
65. The results are shown in Table 24. Complete human antibodies
having human .mu. chain+.lambda. chain and human .gamma. chain
+.lambda. chain, respectively, were confirmed in the sera of the
chimeric mice.
33TABLE 24 Concentrations of Human Antibodies in Chimeric Mice
(ELISA) Chimeric ES clone mouse Chimerism (%) IgM, .lambda. (mg/l)
LH13 CLH13-3 35 51 LH13 CLH13-4 85 32 LH13 CLH13-4 30 27
EXAMPLE 68
[0534] Retention of the Human Chromosome in Offsprings of Human
Chromosome #14 Fragment (Containing Antibody Heavy-Chain
Gene)-Transferred ES Cell-Derived Chimeric Mice
[0535] (1) Isolation of Human-Mouse Hybrid Cells Retaining a Human
Chromosome #14 Fragment Containing an Antibody Heavy Chain Gene
[0536] It was observed in Example 42 that a human chromosome #2
fragment transferred into mice was transmitted to their progeny.
Thus, it is expected that the possibility of transmission of human
chromosome #14 to progeny will be increased if a fragment of this
chromosome is used. A9/#14 clone (Example 9; corresponding to
A9/14-C11 clone described in Tomizuka et al., Nature Genet. vol 16,
133-143 (1997)) retaining an intact human chromosome #14 marked
with a G418 resistance gene was subjected to a more detailed FISH
analysis (Example 9). As a result, it was observed that about 10%
of the cell population contained only a very small, fragmented
human chromosome #14. This chromosome fragment is almost of the
same size as the chromosome #2 fragment (Example 12) and believed
to contain the G418 resistance marker.
[0537] In order to isolate the cell clones containing the
fragmented human chromosome #14, (about 300) A9/#14 cells were
seeded on 10 cm plates and cultured. At day 10 of the cultivation,
31 colonies were picked up. Genomic DNAs were prepared from these
clones and subjected to PCR analysis in the same manner as in
Example 9 using chromosome #14 specific primers (the 18 primers
shown in Example 9 were used except PCI and NP). Out of the 16
primers, only IgM, IGG1, IGA2 and IGVH3 were found in one clone
(A9/SC20). Since D14S543 (Science, HUMAN GENETIC MAP (1994); the
base sequence was obtained from databases of GenBank, etc.) which
is a marker located near the human chromosome #14 long arm telomere
was also detected in the clone, the fragment of interest
(hereinafter referred to as "SC20 fragment") retained in the clone
is believed to contain a region adjacent to the chromosome #14
telomere and which contained an antibody heavy-chain gene.
[0538] SC20 fragment was subjected to FISH analysis (Tomizuka et
al., Nature Genet. vol 16, 133-143 (1997)) using a human
chromosome-specific probe. As a result, it was observed that the
size of the chromosome in the clone that hybridized to the probe
was smaller than in the control clone (containing an intact
chromosome #14). Thus, it was confirmed that A9/SC20 contained a
fragment of human chromosome #14.
[0539] Further, in order to examine whether SC20 fragment contained
a human chromosome #14-derived centromere sequence, chromosome
samples from A9/SC20 cells were hybridized to
digoxigenin-11-dUTP-labeled human chromosome #14 or #22-specific
.alpha. satelite DNA (purchased from COSMOBIO) which was used as a
probe, followed by FISH analysis according to the method described
in a reference (Tomizuka et al., Nature Genet. vol 16, 133-143
(1997)). As a result, a signal hybridizing to the above probe was
confirmed. Thus, it has become clear that SC20 fragment contains a
human-derived centromere sequence (FIG. 36).
[0540] (2) Transfer of Human Chromosome #14 (Fragment) into TT2F
Cells and Stable Retention of the Chromosome Therein
[0541] A human chromosome #14 fragment was microcell-transferred
into TT2F cells using A9/SC20 as a chromosome donor cell in the
same manner as in Example 9. As a result of G418 (300 .mu.g/ml)
selection, 5 resistant clones were obtained. These ES cell clones
were subjected to PCR analysis (Example 68, Section 1) and FISH
analysis (using human chromosome-specific probes; Tomizuka et al.,
supra) to confirm the retention of a human chromosome #14 fragment.
The results of the FISH analysis are shown in FIG. 37.
[0542] Stable retention of transferred human chromosomes in mice is
important for efficient expression of the transferred genes and
efficient transmission of the transferred chromosomes to their
progeny. Since selection by addition of drugs is impossible after
an ES cell clone has been injected into host embryos, it is desired
that transferred human chromosomes be retained stably even under
non-selective conditions.
[0543] TT2F(SC20)-21 clone containing SC20 fragment was cultured in
a medium not containing G418 for a long period to examine the
retention of SC20 fragment under this condition.
[0544] Briefly, TT2F(SC20)-21 clone was cultured in a selective
medium (G418: 300 .mu.g/ml) for 1 week and then subcultured in a
non-selective medium for 45 days (subcultured every other day with
8-fold dilution). At day 0, 15, 30 and 45 of the subculture,
300-1000 cells were seeded in six 35 mm plates, three of which
contained the selective medium and the other three non-selective
medium. The cells were cultured in these plates for about 1 week
and then the colonies were counted. Chromosome retention ratio
(A/B) was calculated by dividing the total number of colonies in
the 3 plates under selective conditions (A) by the total number of
colonies in the 3 plates under non-selective conditions (B). For
the purpose of comparison, an experiment was conducted using P-21
clone (Example 40) containing W23 fragment derived from human
chromosome #2 in the same manner as described above (the selective
medium contained 0.75 .mu.g/ml of puromycin). The results are shown
in FIG. 38. The values shown in FIG. 38 are average values from 3
independent experiments. SC20 fragment exhibited a high retention
ratio of 95% or above even after the 45 day cultivation under
non-selective conditions. On the other hand, W23 fragment exhibited
a retention ratio of 14% under identical conditions.
[0545] There have been reports of the transfer of a human Y
chromosome-derived artificial chromosome (containing human
Y-derived centromere) into CHO (hamster fibroblasts), DT40 (chicken
B lymphocytes) and mouse ES cell (Shen et al., Hum. Mol. Genet. 6,
1375-1382). Under non-selective cultivation, the artificial
chromosome was retained stably in CHO and DT40. In mouse ES cells,
however, only the chromosome which accidentally acquired the mouse
centromere as a result of rearrangement was retained stably. From
these results, an opinion was proposed that a human-derived
centromere is unstable in mouse ES cells (Shen et al., supra).
[0546] The results described above show that SC20, though it
contains a human-derived centromere (Example 68, Section 1), is
very stable in mouse ES cells. Since the retention of W23 fragment
(which is also suggested to contain a human-derived centromere)
(Tomizuka et al., supra) in mouse ES cells appeared to be unstable,
it is considered that the stability of human-derived chromosomes in
mouse ES cells varies depending on the type of the chromosome.
[0547] From these results, it was demonstrated that SC20 fragment
is very useful as a vector for transferring a gene into mice.
[0548] (3) Production of Chimeric Mice from the ES Cell Clone
Retaining Human Chromosome #14 (Fragment)
[0549] Cells in the frozen stock of G418 resistant ES cell clone
TT2F(SC20)-21 which was obtained in Example 68, Section 2 and which
was confirmed to retain a human chromosome #14 fragment were
thawed, started up for culture and injected into 8-cell stage
embryos obtained by mating male and female mice of ICR (CREA JAPAN,
INC.); the injection rate was 10-12 cells per embryo. After the
embryos were cultured overnight in the medium for ES cells (see
Example 9) to develop into blastocysts, about 10 of the injected
embryos were transplanted to each side of the uterus of foster
mother ICR mice (CREA JAPAN, INC.; 2.5 days after pseudopregnant
treatment).
[0550] As a result of transplantation of a total of 188 injected
embryos, 22 offspring mice were born. Chimerism in the offsprings
can be determined by the extent of TT2 cell-derived agouti coat
color (dark brown) in the host embryo (ICR)-derived albino coat
color (white). Out of the 22 offsprings, 20 mice were recognized to
have partial agouti coat color, indicating the contribution of the
ES cells. Out of the 20 mice, two were chimeric mice in which their
coat color was complete agouti (i.e. ES cell-derived).
[0551] From these results, it was confirmed that ES cell clone
TT2F(SC20)-21 retaining a human chromosome #2 fragment maintains
the ability to produce chimera, that is, the ability to
differentiate into normal tissues of mice.
[0552] Two 5-week old chimeric mice [derived from TT2F(SC20)-21,
chimerism 100%, C14m-16 and -17] were bled to determine the
concentrations of human antibody IgM and IgG in the sera by ELISA
in the same manner as in Example 14. The results are shown in Table
25.
34TABLE 25 Concentrations of Human Antibody Heavy-Chains in
Chimeric Mice (ELISA) Chimeric mouse Chimerism (%) IgM (mg/l) IgG
(mg/l) C14m-16 100 7.9 1.0 C14m-17 100 6.0 1.3
[0553] Human antibodies IgM and IgG were detected in the sera of
both chimeric mice. The concentrations of these human antibodies
were comparable to the concentrations in the chimeric mice
retaining the larger human chromosome #14 fragment (see Example
14). Thus, it was demonstrated that the human antibody gene
contained in SC20 fragment is functional.
[0554] (4) Confirmation of the Retention of Human Chromosome in the
Progeny of Chimeric Mice Derived from the Mouse ES Cells (TT2F, XO)
Retaining a Human Chromosome #14 Fragment, and Detection and
Quantitative Determination of Human Antibody .mu. Chain and .gamma.
Chain in sera of the Progeny
[0555] Examination was made as to whether ES cell-derived
offsprings would be produced by mating the female chimeric mice
C14m-16 and C14m-17 (both having 100% chimerism in coat color) from
Example 68, Section 3 with male ICR mice. By this mating,
offsprings with agouti coat color should be produced from TT2F cell
(agouti: dominant)-derived oocytes in the chimeric mice fertilized
by male ICR mouse (albino: recessive)-derived sperms, and
offsprings with albino coat color should be produced from
ICR-derived oocytes in the chimeric mice. Actually, all of the
viable offspring mice obtained by this mating (30 in total)
exhibited ES cell-derived agouti coat color, indicating efficient
transmission of ES cells to the germ cell lineage. Genomic DNAs
were prepared from the tails of these offspring mice to examine the
retention of a human chromosome fragment by PCR. PCR amplification
was performed using the three primers (IGVH3, IgM and D14S543) of
which the presence in TT2F(SC20)-21 was confirmed. As a result, the
presence of the three markers detected in TT2F(SC20)-21 was
confirmed in 10 out of the 30 offspring mice (33%). These results
show that TT2F cell clone TT2F(SC20)-21 retaining a human
chromosome #14 fragment differentiated into functional oocytes in
the chimeric mice and that the human chromosome #14 fragment was
transmitted to the F.sub.1 progeny derived from the oocytes.
[0556] Detection and quantitative determination of human antibodies
IgM and IgG in sera were performed on 9 out of the 10 offspring
mice which were confirmed to retain a human chromosome #14
fragment, as described below. About 4-8 week-old mice were bled to
detect human antibody .mu. chain and .gamma. chain by ELISA in the
same manner as in Example 14. As a result, human antibody .mu.
chain and .gamma. chain were detected in the sera of all of the
mice tested (see Table 26). Thus, It was confirmed that the human
antibody heavy chain gene also functions in the F.sub.1 progeny
born by the chimeric mice.
35TABLE 26 Concentrations of Human Antibodies IgM and IgG in
Chimeric Mice (ELISA) Mother Mouse Chimeric Individual Mouse No.
IgM (mg/l) IgG (mg/l) C14m-16 16-5 12.9 2.2 C14m-16 16-14 3.5 2.2
C14m-16 16-16 4.1 2.0 C14m-16 16-17 5.5 3.9 C14m-17 17-7 5.7 1.0
C14m-17 17-8 3.6 1.2 C14m-17 17-19 3.5 0.75 C14m-17 17-22 2.4 1.4
C14m-17 17-23 5.3 1.9
[0557] Further, 3 male mice and 4 female mice in the F.sub.1
progeny were mated with MCH(ICR) mice (purchased from CREA JAPAN,
INC.) to obtain F.sub.2 progenies, which were subjected to PCR
analysis of tail DNA and analysis for human antibody .mu. chain
expression as described above. As a result, it was confirmed that
SC20 fragment was transmitted to 30% of the F.sub.2 progeny through
F.sub.1 male mice (43 out of the 142 offsprings were positive) and
to 33% of the F.sub.2 progeny through F.sub.1 female mice (20 out
of the 60 offsprings were positive).
[0558] These results show that a mouse strain was established which
retains the human chromosome #14 fragment (containing a human
antibody heavy chain gene), which expresses human antibody
heavy-chains and which can transmit the human chromosome to the
subsequent generation.
[0559] (5) Stable Retention of a Human Chromosome #14 Fragment in
Mice
[0560] Three F.sub.1 mice (16-5, 17-8 and 17-23 shown in Table 26)
which were obtained in Example 68, Section 4 and which retained
SC20 fragment were used in analysis for the ratio of retention of
SC20 fragment in mice. The mice were injected intraperitoneally
with 0.3 ml of CORCEMID (100 .mu.g/ml) and then killed by
dislocation of the cervical vertebrae in an euthanasic manner,
followed by removal of the brain, liver, spleen, testis and bone
marrow. All of these tissues except the bone marrow were washed
with PBS(-), cut into pieces with scissors for anatomy, given
hypotonic treatment with KCl (0.075 M) for 15 minutes, and fixed in
Carnoy's fixative. Specimens were prepared using the supernatant of
the Carnoy fixation by conventional methods. FISH analysis was
performed using a human chromosome-specific probe (Human COT-1 DNA)
according to the method described in a reference (Tomizuka et al.,
Nature Genetics, 16, 133-143). As to the brain, spleen, liver and
bone marrow, 30 or more nuclei in interphase were selected randomly
for each of these tissues. Then, the number of nuclei in which a
signal was detected (mark "+" in FIG. 39) and the number of nuclei
in which a signal was not detected (mark "-" in FIG. 39) were
counted to calculate the retention ratio. The testis were
classified into the 1st meiosis phase spreads, the 2nd meiosis
phase spreads and sperms. Ten or more spreads or sperms were
selected for each group and then counting was performed in the same
manner as described above to calculate the retention ratio. As a
result, all of the 3 mice exhibited a retention ratio of almost
100% in the brain and liver. A decrease in the retention ratio was
observed in the bone marrow and spleen. In the testis, a retention
ratio of 80-100% was obtained for the 1st meiosis phase spreads,
and a retention ratio of 30-50% for sperms. Assuming that SC20
fragment is retained stably, the theoretical retention ratio should
be 100% for the 1st meiosis phase spreads and 50% for the 2nd
meiosis phase spreads and sperms. Thus, it is believed that SC20
fragment is retained stably in the testis.
[0561] At the same time, fibroblasts were prepared from the tail
and then the ratio of retention of SC20 fragment was examined in
the same manner as in Example 79. As a result, the retention ratios
in mice 16-5, 17-8 and 17-23 were 98%, 96% and 98%, respectively
(50 nuclear plate were tested for each mouse).
[0562] (6) Hereditary Relief of Antibody Production
Ability-Deficient Mice by the Transfer of a Human Chromosome #14
Fragment (Containing Antibody Heavy-Chain Gene)
[0563] The knockout mouse whose antibody .mu. chain gene essential
for the generation of B lymphocytes is disrupted (Section 1,
Example 67) cannot produce antibody because the mouse is deficient
in mature B lymphocytes responsible for humoral immunity. The
following experiment was conducted to examine as to whether this
deficiency could be relieved by transferring SC20 fragment
(containing a human antibody heavy-chain gene) by mating.
[0564] Those mice exhibiting agouti coat color among the progeny
obtained by mating the endogenous antibody heavy-chain one
allele-disrupted TT2F cell clone-derived chimeric mice from Example
49 with MCH(ICR) mice were subjected to Southern blot analysis to
select mice retaining the disrupted allele. A female antibody
heavy-chain-deficient heterozygote thus selected was mated with a
male F.sub.1 offspring (17-7) which was obtained in Example 68,
Section 4 and which retains SC20 fragment. The resultant 5
offspring mice were subjected to both PCR analysis for confirming
the retention of SC20 fragment and determination of human antibody
.mu. chain and .gamma. chain in the sera (see Example 68, Section
4). As a result, it was confirmed that three mice #2, #3 and #5
retained SC20 fragment (Table 27). Furthermore, as a result of the
analysis for mouse antibody .mu. chain expression (Example 75), it
was demonstrated that mice #2 and #3 are mouse .mu. chain-negative,
that is, endogenous antibody heavy-chain-deficient homozygotes
(Table 27). These results were consistent with the results of
Southern blot analysis (see Example 49) using the DNAs prepared
from the tails of the 5 mice. Compared to mouse #1 in which neither
mouse nor human antibody heavy chain was detected, very high
concentrations of human antibody .mu. chain (310 mg/l) and .gamma.
chain (860 mg/l) were detected in mouse #3 which is antibody
heavy-chain-deficient homozygote and which retains the human
chromosome #14 fragment. Further, quantitative determination of
human .gamma. subclasses was performed on mouse #3 in the same
manner as in Example 29 to detect all of the 4 subclasses
(.gamma.1, .gamma.2, .gamma.3 and .gamma.4). In particular, the
concentration of human .mu. chain in this mouse is comparable to
the concentration of mouse .mu. chain in wild-type mice (Mendez et
al., Nature Genet. 15, 146-156 (1977)). These results show that the
symptom of inability for antibody production because of desruption
of endogenous heavy-chain gene (deficiency of B lymphocytes: see
Kitamura et al., Nature, 350, 423-, 1991) in this mouse was cured
by the transfer of human chromosome #14 fragment SC20 (containing
an antibody heavy-chain gene), and that the mouse has recovered the
ability to produce antibody and the ability to produce B
lymphocytes.
36TABLE 27 Mouse Retention of Mouse .mu. Human IgM Human IgG No.
SC20 Fragment Chain (mg/l) (mg/l) 1 - - Below detec- 0.33 tion
limit 2 + + 8.4 5.3 3 + - 310 860 4 - + Not Not measured measured 5
+ + 4.8 0.86
EXAMPLE 69
[0565] Retention of the Human Chromosome in Offsprings of Human
Chromosome #22 (Fragment)-Transferred ES Cell-Derived Chimeric
Mice
[0566] (1) Fragmentation of Human Chromosome #22 using Microcell
Fusion
[0567] Since it was observed that both a human chromosome #2
fragment (Example 42) and a human chromosome #14 fragment (Example
68, Section 4) once transferred into mice were transmitted to their
offsprings, it is expected that fragmentation of human chromosome
#22 would increase the possibility of transmission of this
chromosome to offspring mice. When a human chromosome is
transferred into a recipient cell by microcell fusion, it is
observed that 40-80% of the transferred clones retain the human
chromosome which has been fragmented at the time of fusion
(Oshimura et al., Protein, Nucleic Acid, Enzyme, vol. 35, No. 14,
1990). The present inventors tried to fragment human chromosome #22
utilizing this phenomenon.
[0568] A microcell fusion experiment (see Example 1) was conducted
using clone 6-1 from Example 35 as a chromosome donor cell and
wild-type mouse A9 cells as a recipient cell, thereby producing
seventy-three G418 resistant clones. Genomic DNAs were prepared
from the resultant clones and then screened by PCR using Ig A
primers (Example 2). Sixty-seven clones which retained human IgA
gene were subjected to PCR analysis using primers specific to 8
markers located on human chromosome #22 (D22S315, D22S275, D22S280,
D22S278, D22S283, D22S272, D22S282 and D22S274; for the order of
location on human chromosome #22, see Nature, vol. 377, 367-379
(1995); base sequences for these primers were obtained from
databases of such as GenBank). As a result, it was found that a
part of the markers disappeared in 25 clones. Thus, it was
suggested that chromosome #22 was fragmented in these clones (FIG.
40). Among them, clone #22 and clone #28 are considered to have a
fairly small fragment, because markers other than Ig .lambda. and
D22S315 disappeared in the former and markers other than Ig.lambda.
disappeared in the latter (FIG. 40). Clone #28 was subjected to
FISH analysis (see Example 18) using a human chromosome-specific
probe. The results are shown in FIG. 41. It is observed that the
size of the chromosome hybridizing to the probe is smaller in this
clone than in the control clone (containing an intact chromosome
#22). Thus, a human chromosome #22 fragment containing antibody
.lambda. gene could be obtained as a result of fragmentation which
occurred at the time of microcell fusion.
[0569] (2) Transfer of Chromosome #22 (Fragment) into TT2F
Cells
[0570] Chromosome #22 (fragment) was microcell-transferred into
TT2F cells by the method described in Example 2, using clones #22,
#28 and 6-1 as chromosome donor cells. Clones #22 and A9/#22(6-1)
were subjected to puromycin (0.75 .mu.g/ml) selection, and clone
#28 was subjected to G418 (225 .mu.g/ml) selection. As a result,
drug resistant clones were obtained as follows: 13 from clone #22,
5 from clone 6-1 and 3 from clone #28. These ES cell clones are
subjected to PCR analysis and FISH analysis to confirm the
retention of human chromosome #22 (fragment) in the same manner as
in Example 69, Section 1.
[0571] (3) Production of Chimeric Mice from ES Cell Clones
Retaining Human Chromosome #22 (Fragment)
[0572] In the same manner as in Example 3, chimeric mice are
produced from the drug resistant ES cell clones which were obtained
in Example 69, Section 2 and which were confirmed to retain human
chromosome #22. Confirmation of the retention of human chromosome
#22 (fragment) in the resultant chimeric mice is performed by the
method described in Example
[0573] (4) Transmission of Human Chromosome #22 (Fragment) to
Offsprings
[0574] The chimeric mice retaining human chromosome #22 (fragment)
are mixed and mated with ICR mice. Retention of a human chromosome
#22 fragment in the offsprings is examined by PCR using genomic
DNAs prepared from the tails of the offspring mice having agouti
coat color (see Examples 30, 42 and 43). As shown in Examples 42
and 43, mouse ES cell clones retaining human chromosome #22 or a
fragment thereof can differentiate into oocytes or sperms
functional in chimeric mice, thereby allowing to the human
chromosome #22 (fragment) to be transmitted their progenies. Thus,
it is possible to establish a mouse strain which retains human
chromosome #22 (fragment) containing human antibody
light-chain.lambda. gene and which can transmit it to the
subsequent generation.
EXAMPLE 70
[0575] Production of Mice Retaining Both Human Chromosome #2
(Fragment) and #14 (Fragment) by Mating
[0576] The human chromosome #2 (fragment)-retaining mouse strain
from Example 42 or 43 is mated with the human chromosome #14
(fragment)-retaining mouse strain from Example 68 to produce
offsprings. Genomic DNAs are prepared from the tails of the
offspring mice. The DNA is analyzed by PCR, etc. (Examples 9, 42
and 43) to produce those mice which retain both human chromosome #2
partial fragment and human chromosome #14 (fragment).
EXAMPLE 71
[0577] Production of Mice Retaining Both Human Chromosome #22
(Fragment) and #14 (Fragment) by Mating
[0578] The human chromosome #22 (fragment)-retaining mouse strain
from Example 69 is mated with the human chromosome #14
(fragment)-retaining mouse strain from Example 68 to produce
offsprings. Genomic DNAs are prepared from the tails of the
offspring mice. The DNA is analyzed by PCR, etc. (Examples 30, 42
and 43) to produce those mice which retain both human chromosome
#22 (fragment) and #14 (fragment).
EXAMPLE 72
[0579] Production of Mice Retaining the Three Human Chromosomes #2
(Fragment), #14 (Fragment) and #22 (Fragment) by Mating
[0580] The mouse strain retaining both human chromosome #2
(fragment) and #14 (fragment) obtained in Example 71 is mated with
the mouse strain retaining a human chromosome #2 fragment obtained
in Example 42 or 43 to produce offsprings. Genomic DNAs are
prepared from the tails of the offspring mice. The DNA is analyzed
by PCR, etc. (Examples 9, 30, 42 and 43) to produce those mice
which retain all of the three human chromosomes, #22 (fragment),
#14 (fragment) and #2 (fragment). Alternatively, mice retaining all
of the above three human chromosomes may also be obtained by mating
the mouse strain retaining both human chromosome #2 (fragment) and
#14 (fragment) from Example 70, with the mouse strain retaining a
human chromosome #22 fragment from Example 69.
EXAMPLE 73
[0581] Production of a Complete Human Antibody-Producing Mouse
Strain by Mating
[0582] The mouse strains retaining human chromosomes #2+#14
(Example 70), #14+#22 (Example 71) and #2+#14+#22 (Example 72),
respectively, are repeatedly mated with a mouse strain deficient in
endogenous antibody heavy-chain and light-chain .kappa. genes. From
the resultant offsprings, those mouse strains which retain human
chromosomes #2+#14, #14+#22 or #2+#14+#22 and which are homozygotes
in the deficiency of endogenous antibody heavy-chain and
light-chain .kappa. genes, are selected by PCR analysis, etc.
(Examples 9, 30, 42 and 43). In these strains, complete human
antibodies are mainly produced (Green et al., Nature Genetics, 7:
13-, 1994; Lonberg et al., Nature, 368:856-, 1994).
[0583] Hereinbelow, the establishment of a mouse strain which
retains both a human chromosome #2 fragment and a human chromosome
#14 fragment and which is homozygote in the deficiency of
endogenous antibody heavy-chain and light-chain .kappa. genes will
be described. The 4 strains used for the mating and the method for
assaying the genotypes of each strain are as follows.
[0584] (1) The mouse strain from Example 42 retaining a human
chromosome #2 fragment: the retention of the human chromosome #2
fragment is assayed by PCR analysis of the tail-derived DNA as
described in Example 42 and by the expression of human antibody
.kappa. chain in the sera.
[0585] (2) The mouse strain from Example 68, Section 4 retaining a
human chromosome #14 fragment: the retention of the human
chromosome #14 fragment is assayed by PCR analysis of the
tail-derived DNA as described in Example 68, Section 4 and by the
expression of human antibody .mu. chain in the sera.
[0586] (3) The antibody heavy-chain knockout mouse strain from
Example 67, Section 1: heavy-chain deficiency-homozygotes or
heterozygotes were assayed by Southern blot analysis of the
tail-derived DNA as described in Example 67, Section 1 and by the
presence or absence of the expression of mouse antibody .mu. chain
in the sera (see Example 75).
[0587] (4) The antibody .kappa. chain knockout mouse strain from
Example 80: .kappa. chain deficiency-homozygotes or heterozygotes
were assayed by Southern blot analysis of the tail-derived DNA as
described in Example 80.
[0588] A mouse strain which retains all of the 4 genotypes (i.e.,
retaining a human chromosome #2 fragment, retaining a human
chromosome #14 fragment, antibody heavy-chain-deficiency homozygote
or heterozygote, and antibody .kappa. chain-deficiency homozygote
or heterozygote) was established by mating the above 4 strains with
each other. Specifically, after the above 4 strains used as
starting materials were mated several times, a male mouse having
the genotypes of "retaining the human chromosome #14 fragment,
antibody heavy-chain-deficiency homozygote and antibody .kappa.
chain-deficiency heterozygote" was mated with a female mouse having
the genotypes of "retaining the human chromosome #2 fragment,
antibody heavy-chain-deficiency homozygote and antibody .kappa.
chain-deficiency homozygote" or "retaining the human chromosome #2
fragment, antibody heavy-chain-deficiency homozygote and antibody
.kappa. chain-deficiency heterozygote" or "retaining the human
chromosome #2 fragment, antibody heavy-chain-deficiency
heterozygote and antibody .kappa. chain-deficiency homozygote". As
a result, mouse HK23 "retaining the human chromosome #2 fragment,
retaining the human chromosome #14 fragment, antibody
heavy-chain-deficiency homozygote and antibody .kappa.
chain-deficiency heterozygote" and mouse HK29 "retaining the human
chromosome #2 fragment, retaining the human chromosome #14
fragment, antibody heavy-chain-deficiency heterozygote or
wild-type, and antibody .kappa. chain-deficiency heterozygote" were
obtained. FIG. 42 shows the concentration of each antibody in the
sera and the genotypes of these mice, together with the data on
mouse HK28 which was also produced by the above-described mating
and which has the genotypes of "retaining the human chromosome #2
fragment, retaining the human chromosome #14 fragment, antibody
heavy-chain-deficiency heterozygote or wild-type, and antibody
.kappa. chain-deficiency wild-type". A complete human antibody
consisting of human .mu. chain and human .kappa. chain was detected
at a concentration of 18 mg/l in the serum of mouse HK23 (Example
38).
[0589] It is possible to produce those mice having the genotypes of
"retaining the human chromosome #2 fragment, retaining the human
chromosome #14 fragment, antibody heavy-chain-deficiency homozygote
and antibody .kappa. chain-deficiency homozygote" by mating the
mice obtained by the above mating with each other. In this mouse
strain, it is expected that human antibody .kappa. chain will be
expressed at a higher concentration than in mouse HK23 because the
deficiency of the endogenous .kappa. chain gene is substituted by
the human antibody .kappa. chain gene contained in the human
chromosome #2 fragment (Lonberg et al., Nature, 368, 856-, 1994).
It is also expected that the concentration of a complete human
antibody consisting of human heavy-chain and human .kappa. chain
will increase further.
EXAMPLE 74
[0590] Production of a Human Antibody-Producing Hybridoma From a
Mouse Strain which is Obtained by Mating and Which Retains a Human
Chromosome(s) Containing a Human Antibody Gene(s)
[0591] The mice retaining a human chromosome(s) containing a human
antibody gene(s) which were obtained in Example 42, 43, 68, 69, 70,
71, 72 or 73 are immunized with an antigen of interest in the same
manner as in Example 25. The spleen is removed from each mice and
the spleen cells are fused with myeloma cells to produce
hybridomas. After cultivation for 1-3 weeks, the culture
supernatant is analyzed by ELISA. The ELISA is performed by the
method described in Examples 14, 15, 21, 22, 25, 33, 34, 37 and 38.
As a result, human antibody positive clones and clones which are
human antibody positive and specific to the antigen used in the
immunization are obtained.
EXAMPLE 75
[0592] Detection and Determination of Mouse IgM in sera of Chimeric
Mice Derived from the Mouse Antibody Heavy-Chain Both
Alleles-Disrupted TT2F Cell Clone
[0593] Offspring mice were born in the same manner as in Example 40
from the mouse antibody heavy-chain both alleles-disrupted TT2F
cell clone (#131-3) from Example 51. Three mice having chimerisms
of 0%, 50% and 99%, respectively, were selected from the offspring
mice. Mouse IgM in their sera was detected and determined. Briefly,
the chimeric mice of about 2 weeks after birth were bled and mouse
IgM concentration in the sera was determined by ELIZA by the same
procedures as in Example 14. A PBS-diluted anti-mouse IgM antibody
(Kirkegaard & Perry Laboratories Inc., 01-18-03) was fixed, and
then a PBS-diluted serum sample supplemented with 5% FBS was added.
Peroxidase-labeled anti-mouse IgM antibody (Kirkegaard & Perry
Laboratories Inc., 074-1803) was added and the absorbance at 450 nm
was determined using TMBZ as a substrate. Purified mouse IgM
(Pharmingen, 0308ID) was used as a standard. This standard was
diluted stepwise with FBS-supplemented PBS. The results are shown
in Table 28. Of the chimeric mice derived from the mouse antibody
heavy-chain both alleles-disrupted TT2F cells, the mouse having a
chimerism of 99% exhibited a low mouse IgM concentration. Thus, it
was confirmed that the mouse heavy-chain gene from the ES cells
hardly functions in this mouse.
37TABLE 28 Concentration of Mouse IgM in Chimeric Mice (ELISA)
Chimerism % IgM (mg/l) 0 12 50 11 99 1.5
EXAMPLE 76
[0594] Preparation of a Targeting Vector for Knocking Out Antibody
Light-Chain .kappa. gene in ES Cells
[0595] Plasmid LoxP-PGKPuro in which LoxP sequence was inserted at
both ends of a puromycin resistance gene was prepared in the same
manner as in Example 48, Section 1. Briefly, a puromycin resistance
cassette PGKPuro was cut out from PGKPuro plasmid DNA (Watanabe et
al., Biochem. Biophys. Res. Commun. 213:130-137 (1995); released
from Peter W. Laird, Whitehead Institute for Biochemical Research
and Massachusetts Institute of Technology, Dept. of Biology,
Cambridge, Mass.) using restriction enzyme SailI and then blunted.
PGKPuro was inserted into the SmaI and EcoRV restriction sites of a
LoxP-sequence containing plasmid to produce plasmid pLoxP-PGKPuro
(FIG. 30). Further, a DNA fragment comprising a genomic DNA
constant region containing the mouse antibody light-chain.kappa. J
region and constant region was replaced with the LoxP-PGKPuro gene
in the same manner as in Example 48 (FIG. 31).
EXAMPLE 77
[0596] Production of an Antibody Light-Chain Gene Both
Alleles-Disrupted Strain Antibody
Light-Chain-Deficient-Heterozygote (and Antibody
Heavy-Chain-Deficient-Homozygote) Mouse ES Cells
[0597] (1) A Puromycin Resistance Gene was Inserted into the
Antibody Light-Chain Deficient-Heterozygote TT2F Clone (HD43)
Obtained in Example 58 to Give a Strain in which Both Alleles of a
Light-Chain Gene were Disrupted.
[0598] The antibody light-chain targeting vector prepared in
Example 76 was linearized with restriction enzyme KpnI to transform
HD43 clone in the same manner as in Example 58. The resultant
transformants were subjected to selective culture at a puromycin
concentration of 0.75 .mu.g/ml. At day 7-9 of the cultivation,
colonies formed were picked up. A part of these colonies was stored
frozen, and the remaining part was used to prepare genomic DNA in
the same manner as in Example 49. Genomic DNAs from the puromycin
resistant strains were digested with restriction enzyme EcoRI
(Takara Shuzo), separated by agarose gel electrophoresis and
subjected to Southern blot analysis to detect homologous
recombinants using the probe described in Example 48, Section 4
(see Examples 58 and 59). As a result, 4 clones in which both
alleles of an antibody light-chain were disrupted were obtained
from the 74 clones analyzed. Under usual culture conditions, no
changes in growth rate and morphology were observed in these
clones, as compared to the TT2F clone before gene disruption. This
suggests that the clones under consideration retain the ability to
produce chimera.
[0599] (2) Production of Chimeric Mice from the Antibody
Heavy-Chain-Deficient-Homozygote and Antibody Light-Chain Gene Both
Alleles-Disrupted Clone
[0600] Cells in the frozen stock of antibody light-chain gene both
alleles-disrupted TT2F cell clone HD43P-10 from Example 77, Section
1 were thawed, started up for culture and injected into 8-cell
stage embryos obtained by mating male and female mice of ICR (CREA
JAPAN, INC.); the injection rate was 10-12 cells per embryo. After
the embryos were cultured overnight in the medium for ES cells (see
Example 9) to develop into blastocysts, about 10 of the injected
embryos were transplanted to each side of the uterus of foster
mother ICR mice (CREA JAPAN, INC.; 2.5 days after pseudopregnant
treatment).
[0601] As a result of transplantation of a total of 161 injected
embryos, 37 offspring mice were born. Chimerism in the offsprings
can be determined by the extent of TT2 cell-derived agouti coat
color (dark brown) in the host embryo (ICR)-derived albino coat
color (white). Out of the 37 offsprings, 9 mice were recognized to
have partial agouti coat color, indicating the contribution of the
ES cells. Out of the 9 mice, four were chimeric mice in which more
than 80% of their coat color was agouti (i.e. ES cell-derived).
[0602] From these results, it was confirmed that antibody
light-chain both alleles-disrupted ES cell clone HD43P-10 maintains
a high ability to produce chimera.
EXAMPLE 78
[0603] Removal of the G418 Resistance and Puromycin Resistance
Marker Genes from the Antibody Light-Chain Deficient-Homozygote
(and Antibody Heavy-Chain Deficient-Homozygote) TT2F Cell Clone
[0604] From the antibody light-chain both alleles-disrupted
HD43P-10 clone (puromycin resistant, G418 resistant) which was
obtained and confirmed to have a high chimera-forming ability in
Example 77, the puromycin resistance and G418 resistance marker
genes were removed by the procedures described in Example 52.
Briefly, an expression vector pBS185 (BRL) containing a Cre
recombinase gene which causes a site-specific recombination between
the LoxP sequences inserted at both ends of the G418 resistance
marker gene was transferred into the clone described above in the
same manner as in Example 52. The resultant puromycin (0.75
.mu.g/ml) sensitive clones (6 clones) were grown to confluence in
35 mm plates in the same manner as in Example 52. Three fifths
(3/5) of the resultant culture were suspended in 0.5 ml of a
preservation medium [ES medium+10% DMSO (Sigma)] and stored frozen
at -80.degree. C. The remaining two fifth (2/5) were divided into
two portions and inoculated into two 12-well gelatin-coated plates.
Cells in one plate were cultured in non-selective medium for 2
days. Cells in other plate were cultured in the presence of 300
.mu.g/ml of G418 for 2 days. As a result, 5 puromycin sensitive and
G418 sensitive clones which would be killed in the presence of G418
were obtained.
EXAMPLE 79
[0605] Increase in the Expression of Human Antibody .kappa. Chain
in sera as a Result of Mating a Human Chromosome #2
Fragment-Retaining Mouse Strain with C57BL/6 Strain
[0606] The hereditary background of the progenies of the chimeric
mice [hereinafter referred to as "F.sub.1(chimera.times.MCH)"]
which were described in Examples 43 and 44 and which retain a human
chromosome #2 fragment (hereinafter referred to as "W23 fragment")
is that they are a mixture of TT2F cell (Example 39)-derived CBA
mouse strain and C57BL/6 mouse strain, and MCH(ICR) mouse strain
mated with the chimeric mice. In order to observe the behavior of
W23 fragment under a hereditary background as homogeneous as
possible, first, F.sub.1(chimera.times.MCH) were back-crossed with
MCH(ICR). The offspring mice obtained by the mating of
F.sub.1(chimera.times.MCH) (randomly selected 8 male and 6 female
mice).times.MCH(ICR) were examined as to whether they would retain
W23 fragment in the same manner as in Example 43. As a result, it
was confirmed that W23 fragment was transmitted through male to 8%
of the offsprings (25 out of the 324 offsprings were positive) and
through female to 22% of the offsprings (32 out of the 148
offsprings were positive). When the resultant
F.sub.2(F.sub.1.times.MCH)(randomly selected 8 male and 8 female
mice) were further mated with MCH(ICR), the transmission ratio was
9% through male (30 out of the 346 offsprings were positive) and
24% through female (48 out of the 202 offsprings were positive).
Thus, the results was similar to that obtained by the mating of
F.sub.1(chimera.times.MCH).times.MCH(ICR).
F.sub.3(F.sub.2.times.MCH) were obtained by the latter mating.
[0607] The concentrations of human antibody .kappa. chain in the
sera of 4-12-week old chimeric mice (FIG. 43, indicated as
"Chimera", 4 mice), F.sub.1(chimera.times.MCH) (19 mice),
F.sub.2(F.sub.1.times.MCH) (39 mice) and F.sub.3(F.sub.2.times.MCH)
(33 mice) were determined in the same manner as in Example 44. The
results are shown in FIG. 43. Human antibody .kappa. chain was
detected in all of the mice retaining W23 fragment. On the other
hand, the .kappa. chain concentrations varied greatly in
F.sub.2(F.sub.1.times.MCH) and F.sub.3(F.sub.2.times.MCH); the
averaged values in these groups were lower than those in the
chimeric mice and F.sub.1(chimera.times.MCH).
[0608] In order to examine the influence which would be caused by
the mating with a strain other than MCH(ICR), the same
F.sub.2(F.sub.1.times.MCH) mice as used in the experiment of mating
with MCH(ICR) were mated with C57BL/6N (purchased from CREA JAPAN,
INC.). Concentrations of .kappa. chain were determined in the same
manner on the resultant 26 mice retaining W23 fragment
[F.sub.3(F.sub.2.times.C57BL/6)]- . As a result, these mice
exhibited .kappa. chain concentrations as high as those in the
chimeric mice and F.sub.1(chimera.times.MCH) (FIG. 43). As
described above, F.sub.3(F.sub.2.times.MCH) and
F.sub.3(F.sub.2.times.C57BL/6) are derived from the same
F.sub.2(F.sub.1.times.MCH) mice as one of the parents. Therefore,
it is believed that the difference between
F.sub.3(F.sub.2.times.MCH) and F.sub.3(F.sub.2.times.C57BL/6) is in
their hereditary background alone. Thus, it is indicated that
difference in hereditary background influences the amount of
expression of human antibody chain. Further, it has become clear
that the hereditary background of C57BL/6 is more desirable than
that of MCH(ICR) for efficient expression of human antibody .kappa.
chain. From similar experiments, it has been demonstrated that the
hereditary background of C3H HeN (purchased from CREA JAPAN, INC.)
is comparable to or better than that of C57BL/6 for efficient
expression of human antibody .kappa. chain.
[0609] The following experiment was conducted to examine as to
whether the influence of hereditary background on antibody .kappa.
chain concentrations observed above is related to the ratio of
chromosome retention (stability) at the level of individual mice.
Briefly, metaphase chromosome samples were prepared from
tail-derived fibroblasts and bone marrow cells of 2F-1 mouse (serum
.kappa. chain concentration: 84 mg/l) and 1F-3 mouse (serum .kappa.
chain concentration: 13 mg/l) in F.sub.1(chimera.times.MCH) and
subjected to FISH analysis (Tomizuka et al., Nature Genetics, vol
16, 133-143). The ratio of those metaphase spreads containing W23
fragment hybridizing to a human chromosome-specific probe to all of
the spreads observed was determined. It is believed that the
resultant values represent the W23 fragment retention ratios in
fibroblasts and bone marrow cells, respectively. As a result, with
respect to 2F-1, the retention ratio was 51% in fibroblasts and 34%
in bone marrow cells; and with respect to 1F-3, the retention ratio
was 23% in fibroblasts and 18% in bone marrow cells (more than 50
nuclear plate were measured for each case). These results suggest
that the .kappa. chain concentrations in sera correlated to the
ratios of retentions of W23 fragment in fibroblasts and bone marrow
cells. In other words, it is very likely that hereditary background
influences the stability of the transferred human chromosome
fragment itself. Thus, it is believed that the hereditary
background of C57BL/6 or C3H strain is desirable for efficient
expression of a gene not only on the chromosome #2 fragment
described herein but also on other chromosome fragments (e.g.
chromosome #14 fragment).
[0610] In order to verify the above conjecture, a male mouse #17-7
in F.sub.1(chimera.times.MCH) which retains the human chromosome
#14 fragment obtained in Example 68 (hereinafter referred to as
"SC20 fragment") and which expresses a human antibody heavy-chain
in the serum was mated with MCH(ICR) and C57BL/6. Of the resultant
offsprings, two F.sub.2(F.sub.1.times.MCH) mice and two
F.sub.2(F.sub.1.times.C57BL/6) mice, each retaining SC20 fragment,
were subjected to determination of human antibody heavy-chain
concentrations in the sera (see Example 68). Furthermore, metaphase
chromosome samples were prepared from the tails of these mice and
then the ratio of SC20 fragment retention was determined in the
same manner as for W23 fragment. As a result, the human .mu. chain
concentration was 11.0 mg/l and 1.1 mg/l and the chromosome
retention ratio 74% and 54% in F.sub.2(F.sub.1.times.MCH), whereas
the human .mu. chain concentration was 47 mg/l and 54 mg/l and the
chromosome retention ratio 84% and 88% in
F.sub.2(F.sub.1.times.C57BL/6). F.sub.2(F.sub.1.times.C57BL/6) mice
exhibited higher values in both the human .mu. chain concentration
and the chromosome retention ratio. Thus, it has become clear that
the hereditary background of C57BL/6 is desirable for stable
retention of a transferred human chromosome and for efficient
expression of a gene located thereon, as presumed from the results
obtained on W23 fragment-retaining mice.
EXAMPLE 80
[0611] Production of Chimeric Mice from an Antibody
Heavy-Chain-Deficient and Antibody .kappa. Chain-Homologous
Recombinant ES Cell Clone
[0612] Cells in the frozen stock of antibody heavy-chain-deficient
and antibody .kappa. chain-homologous recombinant ES cell clone
HD43 from Example 58 were thawed, started up for culture and
injected into 8-cell stage embryos obtained by mating male and
female mice of ICR (CREA JAPAN, INC.); the injection rate was 10-12
cells per embryo. After the embryos were cultured overnight in the
medium for ES cells (see Example 9) to develop into blastocysts,
about 10 of the injected embryos were transplanted to each side of
the uterus of foster mother ICR mice (CREA JAPAN, INC.; 2.5 days
after pseudopregnant treatment).
[0613] As a result of transplantation of a total of 314 injected
embryos, 51 offspring mice were born. Chimerism in the offsprings
can be determined by the extent of TT2 cell-derived agouti coat
color (dark brown) in the host embryo (ICR)-derived albino coat
color (white). Out of the 51 offsprings, 26 mice were recognized to
have partial agouti coat color, indicating the contribution of the
ES cells. Out of the 26 mice, two were chimeric female mice in
which 100% of their coat color was agouti (i.e. ES
cell-derived).
[0614] From these results, it was confirmed that antibody
heavy-chain-deficient and antibody light-chain-homologous
recombinant ES cell clone HD43 maintains the ability to produce
chimera. In the female mice exhibiting 100% contribution, it is
highly possible that the ES cells have been differentiated into
functional germ cells (oocytes).
[0615] Examination was made as to whether ES cell-derived
offsprings would be produced by mating the above female chimeric
mice (both having 100% chimerism in coat color) with male ICR mice.
By this mating, offsprings with agouti coat color should be
produced from TT2F cell (agouti: dominant)-derived oocytes in the
chimeric mice fertilized by male ICR mouse (albino:
recessive)-derived sperms, and offsprings with albino coat color
should be produced from ICR-derived oocytes. Actually, all of the
viable offspring mice obtained by one mating for each female mouse
exhibited ES cell-derived agouti coat color. Genomic DNAs were
prepared from the tails of these offspring mice to examine the
presence of an antibody .kappa. chain disrupted allele by Southern
blot analysis (Example 58). As a result, mice having an antibody
.kappa. chain disrupted allele were obtained.
[0616] Twenty-seven offspring mice produced by the mating of the
antibody light-chain deficient-heterozygote male and female mice
were subjected to Southern blot analysis (Example 58). As a result,
antibody light-chain wild-type alleles disappeared and only
disrupted alleles were observed in 7 offspring mice. Hence, these
mice were believed to be antibody light-chain-deficient
homozygotes. FIG. 44 shows the results of detection and
quantitative determination of mouse antibody .kappa. chain and
.lambda. chain in the sera.
[0617] In those mice which were judged to be antibody
light-chain-deficient homozygotes by the Southern blot analysis
(Nos. 4, 6, 14, 22, 24, 25 and 26 in FIG. 44), the concentrations
of .kappa. chain are greatly reduced (the remaining .kappa. chain
appears to be derived from their mother mice). Instead, the
concentrations of .lambda. chain are greatly increased in these
mice. These results are consistent with the reported results of
analysis of the antibody .kappa. chain knockout mouse (Yong-Rui Zou
et al., EMBO J. 12, 811-820 (1993)).
[0618] Thus, an antibody .kappa. chain knockout mouse strain could
be established from antibody .kappa. chain homologous recombinant
ES cell clone HD43.
EXAMPLE 81
[0619] Preparation of a Targeting Vector for Inserting Human
Telomere Sequence into Human Chromosome #22
[0620] Fragmentation of human chromosome #22 on which human
antibody .lambda. chain gene (hereinafter referred to as
"Ig.lambda. gene") was located was attempted by inserting human
telomere sequence by homologous recombination (J. E. Itzhaki et
al., Nature Genet., 2, 283-287, 1992). Specifically, a targeting
vector for inserting human telomere sequence into the LIF locus
located very close to Ig .lambda. gene (on the telomere side) was
prepared.
[0621] Human telomere sequence was synthesized by PCR according to
the method of J. J. Harrington et al. (Nature Genet., 15, 345-355,
1997). The PCR product was purified by agarose gel electrophoresis
and then blunted with DNA Blunting Kit (Takara Shuzo). The blunted
PCR product was inserted into the Eco RV site of pBluescript SK
II(+) (Toyobo) by ligation using DNA Ligation Kit (Takara Shuzo)
(pBS-TEL). This plasmid pBS-TEL was sequenced. As a result, it was
found that the telomere sequence had been inserted in the following
direction: Hind III-(TTAGGG)n-Eco RI.
[0622] Subsequently, the LIF gene region on human chromosome #22 to
be used in the homologous recombination was amplified by PCR as
described below, and then cloned into plasmid PBS-TEL. The
sequences of the primers used in the PCR were as follows.
38 Sense primer: 5'-TCGAACTAGTAGGAGAAGTGAACTTGAGGAGGC3' (SEQ ID NO:
65) Antisense primer: 5'-TCGAACTAGTGATTCAGTGATGCTGTGCAGG- -3' (SEQ
ID NO: 66)
[0623] The PCR reaction mixture was composed of 5 .mu.l of
10.times.LA PCR buffer II (Mg.sup.2+ free) (Takara Shuzo); 5 .mu.l
of 25 mM MgCl.sub.2; 8 .mu.l of dNTP mixture (2.5 mM each) (Takara
Shuzo); 10 pmol of sense primer; 10 pmol of antisense primer; 100
ng of template DNA (HFL1, genomic DNA from primary culture human
fibroblasts); 0.5 .mu.l of LA Taq (5 U/.mu.l) (Takara Shuzo) and
sterile distilled water to make a total volume of 50 .mu.l. All of
the operations for preparing the reaction mixture were carried out
on ice. Then, reaction tubes were placed in the well of a thermal
cycler (PCR System 9600, Perkin-Elmer) preset at 85.degree. C.
After the tubes were heated at 94.degree. C. for 1 minute, 35
cycles of reaction were carried out at 98 .degree. C. for 10
seconds and at 65.degree. C. for 5 minutes. The PCR product was
purified and then digested with Spe I (Spe I site was present in
the primers), followed by insertion into the Spe I site in pBS-TEL.
The plasmid in which the LIF gene had been inserted in a opposite
direction of the human telomere sequence (TTAGGG)n was selected (M.
Giovannini et al., Cytogenet Cell Genet 64, 240-244, 1993)
(pBS-TEL/LIF).
[0624] Subsequently, plasmid pGKpuro containing a puromycin
resistance gene (S. Watanabe et al., Biochem. Biophys. Res. Comm.,
213, 130-137, 1995) was digested with Eco RI and blunted, followed
by insertion of Not I linker. The puromycin resistance gene was cut
out by digesting the resultant plasmid with Not I and then inserted
into the Not I site of pBS-TEL/LIF. The plasmid in which the
direction of transcription of the puromycin resistance gene was the
same as that of the LIF gene was selected (pBS-TEL/LIFPuro, see
FIG. 45). The resultant plasmid was amplified in E. coli DH5,
purified with QUIAGEN column (Funakoshi) and used for transfection
(as described later).
EXAMPLE 82
[0625] Transfer of Human Chromosome #22 into Chicken DT40 Cells
[0626] Mouse A9 cells containing human chromosome #22 marked with a
G418 resistance gene (Tomizuka et al., Nature Genet., vol 16,
133-143, 1997; hereinafter referred to as "A9/#22neo") were
cultured in Dulbecco's modified Eagle's Minimal Essential Medium
(hereinafter referred to as DMEM") supplemented with 10% fetal
bovine serum and G418 (800 .mu.g/ml). Chicken DT40 cells were
cultured in DMEM supplemented with 10% FBS, 1% chicken serum and
10.sup.-4 M 2-mercaptoethanol.
[0627] Microcells were prepared as described below (for details,
see Shimizu et al., "Cell Technology Handbook", published by
Yodosha, p. 127-). A9/#22neo cells were cultured in twelve 25
cm.sup.2 centrifuge flasks (Costar) until the cell concentration
reached about 90% saturation. Then, the medium was exchanged with a
medium (DMEM+20% FBS) supplemented with COLCEMID (0.07 .mu.g/ml;
demecolcine, Wako Pure Chemical Industries, Inc.). The cells were
cultured for another 2.5-3 days to form microcells. Thereafter, the
culture solution was removed from the centrifuge flasks, into which
a solution of cytochalasin B (10 .mu.g/ml, Sigma) prewarmed at
37.degree. C. was filled and centrifuged at 34.degree. C. at 8000
rpm for 1 hour. The microcells were suspended in DMEM and purified
by filtration with filters. After the purification, the microcells
were centrifuged at 1500 rpm for 10 minutes and then suspended in 5
ml of DMEM. DT40 cells ( 2.times.10.sup.7) were centrifuged at 1000
rpm for 5 minutes, washed with DMEM twice and suspended in 5 ml of
DMEM. The microcells prepared above were re-centrifuged at 1500 rpm
for 10 minutes and then, without removal of the supernatant, 5 ml
of the previously prepared DT40 suspension was overlayered gently.
After centrifugation at 1300 rpm for 5 minutes, the supernatant was
removed. The cell pellet was suspended in 2 ml of PHA-P (100
.mu.g/ml, DIFCO) and left to stand in an incubator at 37.degree. C.
under 5% Co.sub.2 for 15 minutes. Then, the suspension was
centrifuged at 1700 rpm for 7 minutes. The supernatant was removed
and the cell pellet was loosened by tapping. To the cell pellet, 1
ml of PEG1500 (polyethylene glycol, Boehringer) was added gently
and the pellet was treated for 1.5-2 minutes under agitation. After
this treatment, 1 ml of DMEM was added over approximately 1 minute.
Then, 3 ml of DMEM was added over approximately 2 minutes.
Thereafter, DMEM was added to make a total volume of 11 ml and the
resultant mixture was mixed gently. The mixture was left to stand
for 10 minutes at room temperature and then centrifuged at 1300 rpm
for 5 minutes. The supernatant was removed. The cells were
suspended in 10 ml of the above-described culture medium and
cultured in .phi. 100 mm plates for 24 hours. Twenty-four hours
later, the medium was exchanged with one supplemented with G418 (1
mg/ml). The resultant culture was dispensed into three 24-well
plates (Sumitomo Bakelite), followed by selective culture for about
2 weeks to isolate G418 resistant clones.
[0628] (1) PCR Analysis
[0629] As a result of the selective culture, about thirty G418
resistant clones were obtained. Genomic DNAs were extracted from
these clones using Puregene DNA Isolation Kit (Gentra System).
Using the genomic DNA as a template, PCR was performed with human
Ig .lambda. gene-specific primers to identify clones having human
chromosome #22 containing Ig.lambda. gene. The Ig.lambda.
gene-specific primers used were as follows.
39 5'-GAGAGTTGCAGAAGGGGTGACT-3' (SEQ ID NO: 67)
5'-GGAGACCACCAAACCCTCCAAA-3' (SEQ ID NO: 68)
[0630] The PCR reaction mixture was composed of 5 .mu.l of
10.times.Ex Taq buffer (Takara Shuzo); 8 .mu.l of dNTP mixture (2.5
mM each) (Takara Shuzo); 10 pmol of each primer; 100 ng of genomic
DNA; 0.5 .mu.l of Ex Taq (5 U/.mu.l ) (Takara Shuzo) and sterile
distilled water to make a total volume of 50 .mu.l. All of the
operations for preparing the reaction mixture were carried out on
ice. Then, reaction tubes were placed in the well of a thermal
cycler (PCR System 9600, Perkin-Elmer) preset at 85.degree. C.
After the tubes were heated at 94.degree. C. for 1 minute, 35
cycles of reaction were carried out at 98.degree. C. for 10
seconds, at 56.degree. C. for 30 seconds and at 72.degree. C. for
30 seconds. As a result, 2 clones having human Ig .lambda. gene
were identified. The presence of polymorphic markers (D22S315,
D22S280, D22S283 and D22S274; Polymorphic STS Primer Pair, BIOS; J.
E. Collins et al., Nature 377 suppl.: 367, 1995) located on human
chromosome #22 were detected in these clones by PCR (FIG. 46). The
PCR conditions were the same as used for the detection of human Ig
.lambda. gene. Mark ".largecircle." indicates that the marker was
detected. Mark "X "indicates that the marker was not detected. The
diagram at the left side shows the location of each marker on
chromosome #22 based on a physical map. From these results, it was
suggested that these 2 clones have a almost intact human chromosome
#22. As to the other clones, although human Ig .lambda. gene was
not detected, some of the polymorphic markers on chromosome #22
described above were detected.
[0631] (2) FISH Analysis
[0632] One of the above 2 clones (clone No. 52-18) was subjected to
FISH analysis to examine how the human chromosome #22 actually
existed in cells. Basic operations such as preparation of
chromosome samples, hybridization and detection were performed
according to Tomizuka et al. (Nature Genet. 16, 133-143, 1997). As
a probe, human COT-1 DNA (labeled with Rhodamine) was used. As a
result of observation of 20-30 spreads, it was confirmed that an
almost intact human chromosome #22 was present independently (FIG.
50). Those stained in red are human chromosome #22.
[0633] From these results of analysis, it was thought that chicken
DT40 cell clone 52-18 (hereinafter referred to as "DT40/#22neo")
has intact human chromosome #22.
EXAMPLE 83
[0634] Targeted Truncation of Human Chromosome #22 in Chicken DT40
Cells
[0635] DT40/#22neo from Example 82 was transfected with plasmid
pBS-TEL/LIFPuro prepared in Example 81 and an attempt was made to
perform targeted truncation of the human chromosome #22 on the LIF
locus.
[0636] DT4O//#22neo cells were cultured under the same conditions
as described in Example 82 in the presence of G418 (1 mg/ml).
10.sup.7 cells were washed with cold PPS once, suspended in 0.5 ml
of PBS and placed on ice. Then, 25-30 .mu.g of pBS-TEL/LIFPuro
linearized with Eco RI was added to the cells, mixed with a
pipette, transferred into an electroporation cuvette (Bio-Rad) and
left to stand in ice for 10 minutes. The cuvette was set in a gene
pulser (Bio-Rad) and then a voltage of 550 V was applied at a
capacitance of 25 .mu.F. After the cuvette was left to stand on ice
for 10 minutes, the cells were transferred into 72 cm.sup.2 culture
flasks containing the above-described medium and cultured for 24
hours. Twenty-four hours later, the medium was exchanged with a
medium supplemented with G418 (1 mg/ml) and puromycin (0.3
.mu.g/ml, Sigma). The resultant culture was dispensed into five to
eight 96-well culture plates, followed by selective culture for
about 2 weeks to isolate resistant clones.
[0637] (1) PCR Analysis
[0638] As a result of the selective culture, about 80 resistant
clones were obtained. Genomic DNAs were extracted from these cells
as described above and subjected to PCR to identify homologous
recombinants in which a human telomere sequence was integrated into
the LIF locus. One of the primers was designed such that its
sequence was complementary to a part of the LIF gene region which
was not contained in the vector (see FIG. 47). The other primer was
designed such that its sequence was complementary to a part of the
puromycin resistance gene which was contained in the vector. The
sequences of the primers are as follows.
40 Puro.1: 5'-GAGCTGCAAGAACTCTTCCTCACG-3' (SEQ ID NO: 69) LIF1:
5'-ATGACTCTAAGGCAGGAACATCTGTACC-3' (SEQ ID NO: 70)
[0639] The PCR reaction mixture was composed of 5 .mu.l of
10.times.LA PCR buffer II (Mg.sup.2+ free) (Takara Shuzo); 5 .mu.l
of 25 mM MgCl.sub.2; 8 .mu.l of dNTP mixture (2.5 mM each) (Takara
Shuzo); 10 pmol of each primer; 100 ng of template DNA; 0.5 .mu.l
of LA Taq (5 U/.mu.l ) (Takara Shuzo) and sterile distilled water
to make a total volume of 50 .mu.l. All of the operations for
preparing the reaction mixture were carried out on ice. Then,
reaction tubes were placed in the well of a thermal cycler (PCR
System 9600, Perkin-Elmer) pre-set at 85.degree. C. After the tubes
were heated at 94.degree. C. for 1 minute, 35 cycles of reaction
were carried out at 98.degree. C. for 10 seconds and at 65.degree.
C. for 10 minutes. A 6.3 kb PCR product as shown in FIG. 47 should
be detected only in the homologous recombinants of interest. As a
result of the PCR, this 6.3 kb band was detected in 8 clones
(homologous recombination ratio: about 10%). When this PCR product
was digested with Sal I, a cut pattern was obtained in exactly the
same way as expected. Thus, it was confirmed that these 8 clones
were homologous recombinants.
[0640] Subsequently, whether or not the truncation occurred as
expected in these 8 clones was examined by PCR detection of the
presence of genes (Ig.lambda., LIF, MB, IL2RB, CYP2D6, DIA1, ECGF1
and ARSA; J. E. Collins et al., Nature 377 suppl:367, 1995) and
polymorphic markers (D22S315, D22S275, D22S280, D22S281, D22S277,
D22S278, D22S283, D22S272, D22S282 and D22S274; J. E. Collins et
al., Nature 377 suppl.:367, 1995) on chromosome #22.
[0641] A part of the primer sequences used is as described below.
The remaining primer sequences were the same as used by Tomizuka et
al. (Nature Genet. 16, 133-143, 1997). The presence of LIF is
evident from the experiment described above.
41 CYP2D6 Sense primer: 5'-CTGCGTGTGTAATCGTGTCC- -3' (SEQ ID NO:71)
Antisense primer: 5'-TCTGCTGTGAGTGAACCTGC-3' (SEQ ID NO:72) ECGF1
Sense primer: 5'-AGGAGGCACCTTGGATAAGC-3' (SEQ ID NO:73) Antisense
primer: 5'-TCACTCTGACCCACGATACAGC-3' (SEQ ID NO:74)
[0642] The PCR reaction mixture was composed of 5 .mu.l of
10.times.Ex Taq buffer (Takara Shuzo); 8 .mu.l of dNTP mixture (2.5
mM each) (Takara Shuzo); 10 pmol of each primer; 100 ng of genomic
DNA; 0.5 .mu.l of Ex Taq (5 U/.mu.l ) (Takara Shuzo) and sterile
distilled water to make a total volume of 50.mu.l. All of the
operations for preparing the reaction mixture were carried out on
ice. Then, reaction tubes were placed in the well of a thermal
cycler (PCR System 9600, Perkin-Elmer) pre-set at 85.degree. C.
After the tubes were heated at 94.degree. C. for 1 minute, 35
cycles of reaction were carried out at 98 .degree. C. for 10
seconds, at 56.degree. C. (65.degree. C. for CYP2D6 and ECGF1) for
30 seconds and at 72.degree. C. for 30 seconds. The results are
shown in FIG. 48. Marks ".largecircle." and "X " have the same
meanings as described above. As is clear from this Figure, none of
the genes and markers located on the telomere side of the LIF locus
into which a human telomere sequence had been integrated were
detected in clones 67, 68, 328 and 343. It is suggested that
truncation by the integration of a telomere sequence did occur as
expected in at least those 4 clones.
[0643] (2) FISH Analysis
[0644] Whether the human chromosome #22 had been actually truncated
or not was examined by FISH analysis. The experimental method was
the same as described above. As probes, human COT1 DNA (labeled
with Rhodamine) and plasmid pGKPuro (labeled with FITC) were used.
By COT1 staining, the human chromosome #22 can be visually checked
for truncation in comparison with DT40/#22neo having intact human
chromosome #22. Furthermore, if the chromosome #22 is truncated as
expected on the LIF locus into which the vector has been
integrated, a signal from the Puro probe should be detected at one
end of the telomere of the chromosome #22 fragment. A part of the
results is shown in FIG. 49. As a result of observation of 20-30
spreads for each clone, a small fragment of human chromosome #22
(red) having a Puro probe-derived signal (yellow green) at one end
of the telomere was surprisingly observed in all of the 8
homologous recombinant clones. As for clones 64, 212, 222 and 305
which were presumed not to have undergone truncation from the
results of the PCR analysis, cells having intact chromosome #22
occupied about 10% of all cells.
[0645] These experimental results show that homologous recombinants
in which a human telomere sequence has been integrated into the LIF
locus can be obtained at an efficiency of about 10% in chicken
DT40/#22neo cells and that truncation of the human chromosome #22
has occurred at the integration site in all of the homologous
recombinants (efficiency 100%).
Sequence CWU 1
1
74 1 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 1 tggaaggtgg ataacgccct 20 2 22 DNA Artificial Sequence
Description of Artificial Sequence Primer 2 tcattctcct ccaacattag
ca 22 3 20 DNA Artificial Sequence Description of Artificial
Sequence Primer 3 gcaatcggtc tgccggaaga 20 4 20 DNA Artificial
Sequence Description of Artificial Sequence Primer 4 ttggatcact
ttggacccag 20 5 20 DNA Artificial Sequence Description of
Artificial Sequence Primer 5 ctctcctgca gggccagtca 20 6 22 DNA
Artificial Sequence Description of Artificial Sequence Primer 6
tgctgatggt gagagtgaac tc 22 7 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 7 agtcagggca ttagcagtgc
20 8 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 8 gctgctgatg gtgagagtga 20 9 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 9 tggtggctga aagctaagaa
20 10 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 10 ccagaagaat ggtgtcatta 20 11 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 11 tccaggttct gcagagcaag
20 12 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 12 tgtagttgga ggccatgtcc 20 13 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 13 ccccacccat gatccagtac
20 14 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 14 gccctcagaa gacgaagcag 20 15 22 DNA Artificial Sequence
Description of Artificial Sequence Primer 15 gagagttgca gaaggggtga
ct 22 16 22 DNA Artificial Sequence Description of Artificial
Sequence Primer 16 ggagaccacc aaaccctcca aa 22 17 20 DNA Artificial
Sequence Description of Artificial Sequence Primer 17 ggctatgggg
acctgggctg 20 18 22 DNA Artificial Sequence Description of
Artificial Sequence Primer 18 cagagacaca ggcacgtaga ag 22 19 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 19
ttaagggtca cccagagact 20 20 20 DNA Artificial Sequence Description
of Artificial Sequence Primer 20 tgtagttgga ggccatgtcc 20 21 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 21
caaaaagtcc aaccctatca 20 22 20 DNA Artificial Sequence Description
of Artificial Sequence Primer 22 gccctcagaa gacgaagcag 20 23 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 23
tcgttcctgt cgaggatgaa 20 24 20 DNA Artificial Sequence Description
of Artificial Sequence Primer 24 tcactccgaa gctgcctttc 20 25 21 DNA
Artificial Sequence Description of Artificial Sequence Primer 25
atgtacagga tgcaactcct g 21 26 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 26 tcatctgtaa atccagcagt
20 27 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 27 gatcccatcg cagctaccgc 20 28 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 28 ttcgccgagt agtcgcacgg
20 29 22 DNA Artificial Sequence Description of Artificial Sequence
Primer 29 gatgaactag tccaggtgag tt 22 30 22 DNA Artificial Sequence
Description of Artificial Sequence Primer 30 ccttttggct tctactcctt
ca 22 31 20 DNA Artificial Sequence Description of Artificial
Sequence Primer 31 atagagggta cccactctgg 20 32 20 DNA Artificial
Sequence Description of Artificial Sequence Primer 32 aaccaggtag
gttgatatgg 20 33 20 DNA Artificial Sequence Description of
Artificial Sequence Primer 33 aagttcctgt gatgtcaagc 20 34 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 34
tcatgagcag attaaacccg 20 35 20 DNA Artificial Sequence Description
of Artificial Sequence Primer 35 tgtgaaggag gaccaggtgt 20 36 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 36
tgtaggggtt gacagtgaca 20 37 20 DNA Artificial Sequence Description
of Artificial Sequence Primer 37 ctgagagatg cctctggtgc 20 38 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 38
ggcggttagt ggggtcttca 20 39 20 DNA Artificial Sequence Description
of Artificial Sequence Primer 39 ggtgtcgtgg aactcaggcg 20 40 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 40
ctggtgcagg acggtgagga 20 41 20 DNA Artificial Sequence Description
of Artificial Sequence Primer 41 gcatcctgac cgtgtccgaa 20 42 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 42
gggtcagtag caggtgccag 20 43 20 DNA Artificial Sequence Description
of Artificial Sequence Primer 43 agtgagataa gcagtggatg 20 44 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 44
gttgtgctac tcccatcact 20 45 21 DNA Artificial Sequence Description
of Artificial Sequence Primer 45 ttgtatttcc aggagaaagt g 21 46 20
DNA Artificial Sequence Description of Artificial Sequence Primer
46 ggagacgagg gggaaaaggg 20 47 27 DNA Artificial Sequence
Description of Artificial Sequence Primer 47 atggactgga cctggaggrt
cytctkc 27 48 27 DNA Artificial Sequence Description of Artificial
Sequence Primer 48 atggagyttg ggctgasctg gstttyt 27 49 27 DNA
Artificial Sequence Description of Artificial Sequence Primer 49
atgrammwac tktgkwbcwy sctyctg 27 50 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 50 cagaggcagt tccagatttc
20 51 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 51 tgggatagaa gttattcagc 20 52 27 DNA Artificial Sequence
Description of Artificial Sequence Primer 52 atggacatgr rrdycchvgy
kcasctt 27 53 28 DNA Artificial Sequence Description of Artificial
Sequence Primer 53 ccaagcttca ggagaaagtg atggagtc 28 54 28 DNA
Artificial Sequence Description of Artificial Sequence Primer 54
ccaagcttag gcagccaacg gccacgct 28 55 28 DNA Artificial Sequence
Description of Artificial Sequence Primer 55 ccaagcttca gaggcagttc
cagatttc 28 56 28 DNA Artificial Sequence Description of Artificial
Sequence Primer 56 gggaattcgg gtagaagtca ctgatcag 28 57 28 DNA
Artificial Sequence Description of Artificial Sequence Primer 57
gggaattcgg gtagaagtca cttatgag 28 58 28 DNA Artificial Sequence
Description of Artificial Sequence Primer 58 gggaattcgg gtagaagtca
cttacgag 28 59 60 DNA Artificial Sequence Description of Artificial
Sequence Synthetic probe 59 accttcatcg tcctcttcct cctgagcctc
ttctacagca ccaccgtcac cctgttcaag 60 60 60 DNA Artificial Sequence
Description of Artificial Sequence Synthetic probe 60 tgatgctgca
ccaactgtat ccatcttccc accatccagt gagcagttaa catctggagg 60 61 22 DNA
Artificial Sequence Description of Artificial Sequence Primer 61
ctggggtgag ccggatgttt tg 22 62 22 DNA Artificial Sequence
Description of Artificial Sequence Primer 62 ccaacccagc tcagcccagt
tc 22 63 36 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide 63 aattcccgcg ggtcgacgga
tccctcgagg gtacca 36 64 36 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 64 gggcgcccag
ctgcctaggg agctcccatg gttcga 36 65 33 DNA Artificial Sequence
Description of Artificial Sequence Primer 65 tcgaactagt aggagaagtg
aacttgagga ggc 33 66 31 DNA Artificial Sequence Description of
Artificial Sequence Primer 66 tcgaactagt gattcagtga tgctgtgcag g 31
67 22 DNA Artificial Sequence Description of Artificial Sequence
Primer 67 gagagttgca gaaggggtga ct 22 68 22 DNA Artificial Sequence
Description of Artificial Sequence Primer 68 ggagaccacc aaaccctcca
aa 22 69 24 DNA Artificial Sequence Description of Artificial
Sequence Primer 69 gagctgcaag aactcttcct cacg 24 70 28 DNA
Artificial Sequence Description of Artificial Sequence Primer 70
atgactctaa ggcaggaaca tctgtacc 28 71 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 71 ctgcgtgtgt aatcgtgtcc
20 72 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 72 tctgctgtga gtgaacctgc 20 73 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 73 aggaggcacc ttggataagc
20 74 22 DNA Artificial Sequence Description of Artificial Sequence
Primer 74 tcactctgac ccacgataca gc 22
* * * * *