U.S. patent application number 10/098276 was filed with the patent office on 2003-01-16 for alpha (1,3) galactosyl transferase negative swine.
This patent application is currently assigned to The General Hospital Corporation. Invention is credited to Baetscher, Manfred W., Gustafsson, Kenth T., Sachs, David H..
Application Number | 20030014770 10/098276 |
Document ID | / |
Family ID | 22859145 |
Filed Date | 2003-01-16 |
United States Patent
Application |
20030014770 |
Kind Code |
A1 |
Gustafsson, Kenth T. ; et
al. |
January 16, 2003 |
Alpha (1,3) galactosyl transferase negative swine
Abstract
Transgenic swine in which the normal expression of .alpha.(1,3)
galactosyltransferase is prevented in at least one organ of tissue
type. The absence or inactivation of this enzyme prevents the
production of carbohydrate moieties having the distinctive terminal
Gal.alpha.1-3Gal.beta.1-4GlcNAc epitope that is a significant
factor in xenogeneic, particularly human, transplant rejection of
swine grafts.
Inventors: |
Gustafsson, Kenth T.;
(Buckinghamshire, GB) ; Sachs, David H.; (Newton,
MA) ; Baetscher, Manfred W.; (Winchester,
MA) |
Correspondence
Address: |
Raymond J. Lillie
CARELLA, BYRNE, BAIN, GILFILLAN,
CECCHI, STEWART & OLSTEIN
Six Becker Farm Road
Roseland
NJ
07068
US
|
Assignee: |
The General Hospital
Corporation
|
Family ID: |
22859145 |
Appl. No.: |
10/098276 |
Filed: |
March 15, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10098276 |
Mar 15, 2002 |
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08929940 |
Sep 15, 1997 |
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6413769 |
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08929940 |
Sep 15, 1997 |
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08716443 |
Sep 16, 1996 |
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08716443 |
Sep 16, 1996 |
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PCT/US95/03940 |
Mar 31, 1995 |
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PCT/US95/03940 |
Mar 31, 1995 |
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08228933 |
Apr 13, 1994 |
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Current U.S.
Class: |
800/17 |
Current CPC
Class: |
C12N 2310/121 20130101;
A01K 2267/025 20130101; A01K 2267/02 20130101; C12N 2310/315
20130101; A61K 2035/122 20130101; C12N 15/8509 20130101; C12N
2517/02 20130101; A01K 2217/05 20130101; C12N 2310/111 20130101;
C12N 2830/52 20130101; C12N 2840/203 20130101; C12Y 204/01228
20130101; A01K 67/0275 20130101; A01K 2217/075 20130101; A01K
67/0276 20130101; A01K 2217/072 20130101; C12N 15/1137 20130101;
A01K 2227/108 20130101; C12N 15/113 20130101; C12N 9/1051
20130101 |
Class at
Publication: |
800/17 |
International
Class: |
A01K 067/027 |
Claims
What is claimed is:
1. A transgenic swine is which the normal expression of the
.alpha.(1,3) galactosyltransferase is prevented in at least one
organ or tissue type.
2. The transgenic swine of claim 1 which has been made to produce a
nucleic acid sequence that binds to and prevents the translation of
mRNA coding for .alpha.(1,3) galactosyltransferase in said
swine.
3. The transgenic swine of claim 2 whose genome had been modified
to include a construct comprising a DNA encoding an antisense RNA
to that portion of the .alpha.(1,3) galactosyltransferase coding
region necessary to prevent or express a functional enzyme operably
linked to a promoter therefore.
4. The transgenic swine of claim 3 wherein the promoter is a strong
non-tissue specifiic constitutive or regulatable promoter.
5. The transgenic swine of claim 3 wherein the promoter is a strong
tissue specific constitutive or regulatable promoter.
6. The transgenic swine of claim 1 which has been made to produce
ribozyme.
7. The transgenic swine of claim 6 which has been modified to
include a construct comprising DNA coding for at least that portion
of a ribozyme necessary to inactivate .alpha.(1,3)
galactosyltransferase operably linked to promoter therefor.
8. The transgenic swine of claim 7 wherein the promoter is a strong
non-tissue specific constitutive or regulatable promoter.
9. The transgenic swine of claim 7 wherein the promoter is a strong
tissue specific constitutive or regulatable promoter.
10. An .alpha.(1,3) galactosyltransferase negative swine grown from
a porcine oocyte whose pronuclear material has been removed and
into which has been introduced a pluripotent porcine embryonic
stemcell that is negative for the expression of .alpha.(1,3)
galactosyltransferase
11. A nucleic acid having a sequence selected from the group
consisting of SEQ. ID. NOS. 1 and 13 through 24.
12. The nucleic acid of claim 11 having the sequence set forth in
SEQ. ID. NO. 1.
13. The nucleic acid of claim 11 wherein the SEQ. ID. NOS. are
selected from the group consisting of SEQ. ID. NOS. 13 through
15.
14. The nucleic acid of claim 11 wherein the SEQ. ID. NOS. are
selected from the group consisting of SEQ. ID. NOS. 16 through
21.
15. The nucleic acid of claim 11 wherein the SEQ. ID. NOS. are
selected from the group consisting of SEQ. ID. NOS. 22 through
24.
16. The transgenic swine of claim 1 in which the normal expression
of the .beta. (1,3) galactosyltransferese gene is prevented by
introduction to said swine of an isogenic DNA-containing targeting
vector.
17. The .alpha.(1,3) galactosyltransferese negative swine of claim
16 grown from a porcine zygote which has been modified by
homologous recombination with an isogenic DNA targeting vector at
the .alpha.(1,3) galactosyltransferese chromosomal locus.
18. The .alpha.(1,3) galactosyltransferese negative swine of claim
17 wherein the isogenic DNA targeting vector comprises at least
about 6 kb of isogenic DNA and a promoterless selectable marker
gene.
Description
[0001] Donor organ shortages have led to hopes that
xenotransplantation could serve as an alternative means of organ
availability. Swine, particularly mini-swine, are an attractive
alternative to non-human primate donors because of potentially
greater availability, the reduced risk of zoonotic infections,
appropriate size of organs and the reduced social and ethical
concerns (Sachs, D. H. et al. 1976. Transplantation 22:559-567;
Auchincloss, H. Jr. 1988. Transplantation 46:1-20). However, one of
the major barriers to xenotransplantation is the phenomenon
described as hyperacute rejection (Busch et al. 1972. Am. J.
Pathology 79:31-57; Auchincloss, H. Jr. 1988. Transplantation
46:1-20). This phenomenon describes a very rapid and severe humoral
rejection, which leads to destruction of the graft within minutes
or hours of the transplant of the donor organ. Hyperacute rejection
is apparently mediated by a complex series of events, including
activation of the complement systems, activation of blood
coagulation proteins, activation of endothelial cells and release
of inflammatory proteins (Busch et al. 1972. Am. J. Pathology
79:31-57; Platt, J. L. 1992. ASAIO Journal 38:8-16). There is an
accumulating body of information that implicates a group of
pre-formed antibodies, the so-called natural antibodies, to be of
fundamental importance in the hyperacute rejection seen in grafts
between species. Species combinations in which the recipients of
grafts have circulating antibodies that can initiate the hyperacute
response to the donor species are described as discordant. Pigs and
humans are one such discordant species combination.
[0002] The hyperacute rejection process is initiated when the
natural antibodies of the recipient bind to cells of the donor
organ (Platt et al. 1990. Transplantation 50:870-822; Platt et al.
1990. Immunology Today 11:450-456). It has been suggested that
porcine N-linked carbohydrates carrying a terminal
Gal.alpha.1-3Gal.beta.1-4GlcNAc structure are the major targets for
anti-swine xenoreactive human natural antibodies (Good et al. 1992.
Transplantation Proceedings 24:559-562; Sandrin et al. 1993. Proc.
Natl. Acad. Sci. USA 90:11391-11395). One major difference between
the glycosylation pattern of swine tissues and human tissues is the
presence of high levels of a terminal
Gal.alpha.1-3Gal.beta.1-4GlcNAc structure on swine cells and
tissues. This structure is expressed at high levels in all lower
mammals investigated, but is poorly expressed on cells and tissues
of Old World monkeys, apes and humans (catarrhines) (Galili, U. and
Swanson, K. 1992. Proc. Natl. Acad. Sci. USA 88:7401-7404; Galili
et al. 1987. Proc. Natl. Acad. Sci. USA. 84:1369-1373). A specific
transferase, UDP-Gal:Gal.beta.1-->4GlcNAc
.alpha.1-->3-galactosyltransferase (EC 2.4.1.151; .alpha.(1,3)
galactosyltransferase) is responsible for the transfer of a
terminal galactose to the terminal galactose residue of
N-acetyllactosamine-type carbohydrate chains and lactosaminoglycans
according to the reaction:
Mn.sup.2+
UDP-Gal+Gal.beta.1-->4GlcNAc-R------>Gal1-->3Gal.alpha.1-->4Gl-
cNAc-R +UDP
[0003] where R may be a glycoprotein or a glycolipid (Blanken, W.
M. and Van den Eijinden, D. H. 1985. J. Biol. Chem.
260:12927-12934). Thus the Gal.alpha.1-3Gal.beta.1-4GlcNAc epitope.
Full length cDNA sequences encoding the murine (Larsen et al. 1989.
Proc. Natl. Acad. Sci. USA. 86:8227-8231) and bovine (Joziasse et
al. 1989. J. Biol. Chem. 264:14290-14297) enzymes have been
determined. In addition, the genomic organization of the murine
a(1,3) galactosyltransferase gene has been established (Joziasse et
al. 1992. J. Biol. Chem. 267:5534-5541). A partial sequence
encoding the 3' region of the porcine .alpha.(1,3)
galactosyltransferase cDNA gene has been determined (Dabkowski et
al. 1993. Transplantation Proceedings. 25:2921) but the full length
sequence has not been reported. The absence of the 5' sequence is
significant for the applications described herein. In contrast to
the lower mammals, humans do not express the .alpha.(1,3)
galactosyltransferase. Furthermore, human sequences homologous to
the murine sequence correspond to a processed pseudogene on
chromosome 12 and an inactivated remnant on chromosome 9 (Shaper et
al. 1992. Genomics 12:613-615).
[0004] In accordance with the invention, swine organs or tissues or
cells that do not express .alpha.(1,3) galactosyltransferase will
not produce carbohydrate moieties containing the distinctive
terminal Gal.alpha.1-3Gal.beta.1-4GlcNAc epitope that is a
significant factor in xenogeneic, particularly human, transplant
rejection of swine grafts. Further in accordance with the
invention, is the aspect of diminishing the production of
.alpha.(1,3) galactosyltransferase to an extent sufficient to
prevent the amount produced from providing carbohydrates with the
Gal.alpha.1-3Gal.beta.1-4GlcNAc epitope from being presented to the
cell surface thereby rendering the transgenic animal, organ,
tissue, cell or cell culture immunogenically tolerable to the
intended recipient without requiring complete .alpha.(1,3)
galactosyltransferase gene suppression.
[0005] One principal aspect of the present invention is that the
inventors have isolated the entire porcine .alpha.(1,3)
galactosyltransferase cDNA gene (SEQ. ID NO. 1). The
identification, isolation and sequencing of the entire cDNA gene,
now particularly providing the sequence of the 5' end is an
important advance because, as described in Example 2, this region
has been identified as the most efficient for antisense targeting.
Moreover, as compared with mouse and bovine homologous sequences
(FIG. 2), this region of the .alpha.(1,3) galactosyltransferase
mRNA appears to deviate extensively between these species making it
extremely unlikely that a use of "cross-species" antisense
constructs would be successful.
[0006] Another principle aspect of this invention related to
genetically altered animals, more specifically transgenic, chimeric
or mosaic swine in which the expression of biologically active
.alpha.(1,3) galactosyltransferase is prevented in at least one
organ, tissue or cell type. Transgenic animals carry a gene which
has been introduced into the germline of the animal, or an ancestor
of the animal, at an early developmental stage. The genetic
alteration in transgenic animals is stably incorporated into the
genome as a result of intentional experimental intervention.
Typically, this results from the addition of exogenous foreign DNA
or novel constructs (Palmiter et al. 1986. Ann. Rev. Genet.
20:465). With the advent of embryonic stem (ES) cells and specific
gene targeting, the definition of transgenesis now includes
specific modification of endogenous gene sequences by direct
experimental manipulation and by stable incorporation of DNA that
codes for effector molecules that modulate the expression of
endogenous genes (Gossler et al. 1986. Proc. Natl. Acad. Sci. USA.
83:9065; Schwarzberg et al. 1989. Science 246:799; Joyner et al.
1989. Nature 338:153).
[0007] One preferred approach for generating a transgenic animal
involves micro-injection of naked DNA into a cell, preferentially
into a pronucleus of an animal at an early embryonic stage (usually
the zygote/one-cell stage). DNA injected as described integrates
into the native genetic material of the embryo, and will faithfully
be replicated together with the chromosomal DNA of the host
organism. This allows the transgene to be passed to all cells of
the developing organism including the germ line. Transgene DNA that
is transmitted to the germ line gives rise to transgenic offspring.
If transmitted in a Mendelian fashion, half of the offspring will
be transgenic. All transgenic animals derived from one founder
animal are referred to as a transgenic line. If the injected
transgene DNA integrates into chromosomal DNA at a stage later than
the one cell embryo not all cells of the organism will be
transgenic, and the animal is referred to as being genetically
mosaic. Genetically mosaic animals can be either germ line
transmitters or non-transmitters. The general approach of
microinjection of heterologous DNA constructs into early embryonic
cells is usually restricted to the generation of dominant effects,
i.e., one allele of the transgene (hemizygous) causes expression of
a phenotype (Palmiter et al. 1986. Ann. Rev. Genetics 20:465.)
[0008] In another preferred approach, animals are genetically
altered by embryonic stem (ES) cell-mediated transgenesis (Gossler
et al. 1986, Proc. Natl. Acad. Sci. USA. 83:9065). ES cell lines
are derived from early embryos, either from the inner cell mass
(ICM) of a blastocyst (an embryo at a relatively early stage of
development) or migrating primordial germ cells (PGC) in the
embryonic gonads. They have the potential to be cultured in vitro
over many passages (i.e. are conditionally immortalized), and they
are pluripotent, or totipotent (i.e. are capable of differentiating
and giving rise to all cell types. ES cells can be introduced into
a recipient blastocyst which is transferred to the uterus of a
foster mother for development to term. A recipient blastocyst
injected with ES cells can develop into a chimeric animal, due to
the contributions from the host embryo and the embryonic stem
cells. ES cells can be transfected with heterologous gene
constructions that may cause either dominant effects, inactivate
whole genes or introduce subtle changes including point mutations.
Subsequent to clonal selection for defined genetic changes, a small
number of ES cells can be reintroduced into recipient embryos
(blastocysts or morulae) where they potentially differentiate into
all tissues of the animal including the germ line and thus, give
rise to stable lines of animals with designed genetic
modifications. Totipotent porcine embryonic stem cells can be
genetically altered to have a heterozygous (+/-) mutant, preferably
null mutant allele, particularly one produced by homologous
recombination in such embryonic stem cells. Alternatively, gene
targeting events by homologous recombination can be carried out at
the same locus in two consecutive rounds yielding clones of cells
that result in a homozygous (-/-) mutant, preferably a null mutant
(Ramirez-Solis et al. 1993. Methods in Enzymol. 225:855).
[0009] In one preferred embodiment of this invention a DNA sequence
is integrated into the native genetic material of the swine and
produces antisense RNA that binds to and prevents the translation
of the native mRNA encoding .alpha.(1,3) galactosyltransferase in
the transgenic swine.
[0010] In a particularly preferred embodiment the genome of the
transgenic swine is modified to include a construct comprising a
DNA complementary to that portion of the .alpha.(1,3)
galactosyltransferase coding region that will prevent expression of
all or part of the biologically active enzyme. As the term is used
"integrated antisense sequence" means a non-native nucleic acid
sequence integrated into the genetic material of a cell that is
transcribed (constitutively or inducibly) to produce an mRNA that
is complementary to and capable of binding with an mRNA produced by
the genetic material of the cell so as to regulate or inhibit the
expression thereof.
[0011] In another embodiment of the invention, cells or cell lines
from non-mutant swine are made with the .alpha.(1,3)
galactosyltransferase inactivated on one or both alleles through
the use of an integrated antisense sequence which binds to and
prevents the translation of the native mRNA encoding the
.alpha.(1,3) galactosyltransferase in said cells or cell lines. The
integrated antisense sequence, such as the RNA sequence transcribed
in Example 3 is delivered to the cells by various means such as
electroporation, retroviral transduction or lipofection.
[0012] In another preferred embodiment, the transgenic swine is
made to produce a ribozyme (catalytic RNA) that cleaves the
.alpha.(1,3) galactosyltransferase mRNA with specificity. Ribozymes
are specific domains of RNA which have enzymatic activity, either
acting as an enzyme on other RNA molecules or acting
intramolecularly in reactions such as self-splicing or
self-cleaving (Long, D. M. and Uhlenbeck, O. C. 1993. FASEB
Journal. 7:25-30). Certain ribozymes contain a small structural
domain generally of only about 30 nucleotides called a
"hammerhead". The hammerhead is a loop of RNA that is flanked by
two linear domains that are specific complements to domains on the
substrate to be cleaved. The site on the hammerhead ribozyme that
effects the cleavage of substrate is the base of the stem loop or
hammerhead. As shown in FIG. 3, the ribozymes of the present
invention have flanking sequences complementary to domains near the
5' end of the .alpha.(1,3) galactosyltransferase cDNA gene.
[0013] The DNA for the ribozymes is integrated into the genetic
material of an animal, tissue or cell and is transcribed
(constitutively or inducibly) to produce a ribozyme which is
capable of selectively binding with and cleaving the .alpha.(1,3)
galactosyltransferase mRNA. As it is a catalytic molecule, each
such ribozyme is capable of cleaving multiple substrate
molecules.
[0014] The catalytic "stem loop" of the ribozyme is flanked by
sequences complementary to regions of the .alpha.(1,3)
galactosyltransferase mRNA. In a particularly preferred embodiment
the transgenic swine is modified to integrate a construct
comprising the DNA coding for that portion of catalytic RNA
necessary to inactivate the mRNA of the .alpha.(1,3)
galactosyltransferase operably linked to a promoter therefor.
[0015] In another embodiment of the invention, cells or cell lines
from non-mutant swine are made with the .alpha.(1,3)
galactosyltransferase inactivated on one or both alleles through
the use of an integrated ribozyme sequence which binds to and
cleaves the native mRNA encoding the .alpha.(1,3)
galactosyltransferase in said cells or cell lines. The integrated
ribozyme sequence, such as the RNA sequence transcribed in Example
4 is delivered to the cells by various means such as
electroporation, retroviral transduction or lipofection.
[0016] In another preferred embodiment, using cultured porcine
embryonic stem cells, a mutation, preferably a null mutation is
introduced by gene targeting at the native genomic locus enconding
.alpha.(1.3) galactosvltranferase. Gene targeting by homologous
recombination in ES cells is performed using constructs containing
extensive sequence homology to the native gene, but specific
mutations at positions in the gene which are critical for
generating a biologically active protein. Therefore, mutations can
be located in regions important for either translation,
transcription or those ES clone that have homologously recombined a
gene targeting construct, also termed gene "knock out" construct,
can be achieved using specific marker genes. The standard procedure
is to use a combination of two drug selectable markers including
one for positive selection (survival in the present of drug, if
marker is expressed) and one for negative selection (killing in the
presence of the drug, if marker is expressed) (Mansour et al.,
1988. Nature 336:348) One preferred type of targeting vector
includes the neomycin phosphotransferase (neo) gene for positive
selection in the drug G418, as well as the Herpes Simplex
Virus-thymidine kinase (HSV-tk) gene for selective killing in
gancyclovir Drug selection in G418 and gancyclovir, also termed
positive negative selection (PNS) (Mansour et al. 1988. Nature
336:348; Tubulewicz et al. 1991. Cell 65:1153) allows for
enrichment of ES cell clones that have undergone gene targeting,
rather than random integration events. Confirmation of homologous
recombination events is performed using Southern analysis.
[0017] The design of the .alpha.(1,3) galactosyltransferase
targeting construct is described in Example 6. The procedure as
applied here uses a positive selection (survival) based on
integration of the neo (neomycin resistance), preferably in inverse
orientation to the endogenous .alpha.(1,3) galactosyltransferase
gene locus in a cassette with the phosphoglycerate kinase (PGK-1)
promoter and with flanking oligonucleotides complementary to two
separate regions of the .alpha.(1,3) galactosyltransferase gene
sequence. It is understood that other positive selectable markers
may be used instead of neo. The neo gene is linked with its
promoter to be under control thereof. Downstream from the second
flanking sequence is the HSV-tk gene which, if integrated into the
genome encodes for production of thymidime kinase making the cell
susceptible to killing by gancyclovir (negative selection). The
integration of the neo gene but not the HSV-tk gene occurs only
where integration into the .alpha.(1,3) galactosyltransferase gene
has occurred and provides for both positive and negative selection
of the cells so transformed.
[0018] In another preferred embodiment, using isogenic DNA, it has
become possible to achieve high frequency homologous recombination
even in biological systems, such as zygotes, which do not lend
themselves to the use of elaborate selection protocols and were,
therefore, previously not suitable candidates for the isolation of
cells which showed positive marker attributes indicating homologous
recombination. This use of targeting vectors which include isogenic
DNA which is substantially identical to that of chromosomal
segments of the target recipient cell, can be used to target
zygotes which thereafter develop into transgenic animals. The use
of these zygote cells is preferably to produce a mutation,
preferably a null mutation, at the chromosomal locus encoding
.alpha.(1,3) galactosyltransferese. Thus, these vectors contain
extensive sequence homology to the native gene, but also contain
specific mutations at segments in the gene which are critical for
generating a biologically active protein. Therefore, mutations can
be located in regions important for either translation,
transcription, or those coating for functional domains of the
protein. The high percentage of homologous recombination achieved
using isogenic DNA makes it possible to avoid the need for
selection of clones that have homologously recombined the gene
targeting construct, as described above with respect to the "knock
out" embodiment thereby making it possible to avoid the need for
the standard selection procedure described above.
[0019] More particularly, in this embodiment, PCR is used to
identify and extract 1-2 kb DNA fragments, which are then subjected
to restriction fragment length polymorphism digestions to identify
areas or alleles that are most abundantly present in the line of
mini-swine selected for zygote injection. Known insertion vectors
are used, however, replacement vectors also described in the art
could also be used. It is also possible to use DNA sequences with
an isogenic replacement vector that require only a few kilobases of
uninterrupted isogenic DNA. As such, it is possible to effect
highly efficient homologous recombination such that it is not
necessary to screen targeting vector recombined zygotes. Rather,
genomic DNA screening using PCR can be done after the piglets are
born. These transgenic founder animals, which are observed to have
the transgene targeted to the native .alpha.(1,3)
galactosyltransferese locus (i.e. that are heterozygous null mutant
for that gene, are grown and interbred to produce animals that are
homozygous null mutant for this locus, resulting in the knock out
of the .alpha.(1,3) galactosyltransferese gene which can be
confirmed by antibody or lectin binding assays.
[0020] The swine is preferably an .alpha.(1,3)
galactosyltransferase negative swine grown from a porcine oocyte
whose pronuclear material has been removed and into which has been
introduced a totipotent porcine embryonic stem cell using protocols
for nuclear transfer (Prather et al. 1989, Biol. Reprod. 41:414) ES
cells used for nuclear transfer are negative for the expression of
.alpha.(1,3) galactosyltransferase, or alternatively, totipotent ES
cells used for nuclear transfer are mutated in a targeted fashion
in at least one allele of the .alpha.(1,3) galactosyltransferase
gene.
[0021] The swine is preferably lacking expression of the
.alpha.(1,3) galactosyltransferase gene and bred from chimeric
animals which were generated from ES cells by blastocyst injection
or morula aggregation. ES cells used to generate the preferably
null-mutated chimeric animal were mutated at least in one allele of
the .alpha.(1,3) galactosyltransferase gene locus, using gene
targeting by homologous recombination.
[0022] A chimeric swine is preferably constituted by ES cells
mutated in one allele of the .alpha.(1,3) galactosyltransferase
gene. Derived from mutated ES cells are also germ cells, male or
female gametes that allow the mutation to be passed to offspring,
and allow for breeding of heterozygous mutant sibling pigs to yield
animals homozygous mutant at the .alpha.(1,3) galactosyltransferase
locus. Also described is a swine, deficient for an .alpha.(1,3)
galactosyltransferase protein (i.e., characterized by lack of
expression of .alpha.(1,3) galactosyltransferase protein) and have
little, if any, functional Gal.alpha.1-3Gal.beta.1-4Glc- NAc
epitope-containing carbohydrate antigen on the cell surface are
produced. Further described are methods of producing transgenic
swine and methods of producing tissue from heterozygous swine or
homozygous swine of the present invention. The present invention
also relates to cell lines, such as swine cell lines, in which the
.alpha.(1,3) galactosyltransferase gene is inactivated on one or
both alleles and use of such cell lines as a source of tissue and
cells for transplantation.
[0023] Tissues, organs and purified or substantially pure cells
obtained from transgenic swine, more specifically from hemizygous,
heterozygous or homozygous mutant animals of the present invention
can be used for xenogeneic transplantation into other mammals
including humans in which tissues, organs or cells are needed. The
.alpha.(1,3) galactosyltransferase inactive cells can themselves be
the treatment or therapeutic/clinical product. For example,
keratinocytes rendered .alpha.(1,3) galactosyltransferase inactive
can be used for macular degeneration and pancreatic cells rendered
.alpha.(1,3) galactosyltransferase deficient can be used to replace
or restore pancreatic products and functions to a recipient. In
another embodiment, .alpha.(1,3) galactosyltransferase inactive
cells produced by the present method are further manipulated, using
known methods, to introduce a gene or genes of interest, which
encode(s) a product(s), such as a therapeutic product, to be
provided to a recipient. In this embodiment, the .alpha.(1,3)
galactosyltransferase deficient tissue, organ or cells serve as a
delivery vehicle for the encoded product(s). For example,
.alpha.(1,3) galactosyltransferase deficient cells, such as
fibroblasts or endothelial cells, can be transfected with a gene
encoding a therapeutic product, such as cytokines that augment
donor tissue engraftment, Factor VIII, Factor IX, erythropoietin,
insulin, human major histocompatibility (MHC) molecules or growth
hormone, and introduced into an individual in need of the encoded
product.
[0024] Alternatively, recipient blastocysts are injected or morulae
are aggregated with totipotent embryonic stem cells yielding
chimeric swine containing at least one allele of a mutated,
preferably null-mutated .alpha.(1,3) galactosyltransferase gene
produced by homologous recombination. A chimeric swine is
preferably constituted by ES cells mutated in one allele of the
.alpha.(1,3) galactosyltransferase gene. Derived from mutated ES
cells are also germ cells that allow the mutation to be passed to
offspring, and breeding of heterozygous mutant sibling pigs to
yield animals homozygous mutant at the .alpha.(1,3)
galactosyltransferase locus. Also described is a swine, deficient
for an .alpha.(1,3) galactosyltransferase protein (i.e.,
characterized by essentially no expression of .alpha.(1,3)
galactosyltransferase protein) and with little, if any, functional
Gal.alpha.1-3Gal.beta.1-4GlcNAc epitope-containing carbohydrate
antigen on the cell surface are produced. Further described are
methods of producing transgenic swine and methods of producing
tissue from heterozygous swine or homozygous swine of the present
invention. The present invention also related to cell lines, such
as swine cell lines, in which the .alpha.(1,3)
galactosyltransferase gene is inactivated on one or both alleles
and use of such cell lines as a source of tissue, organs and cells
for transplantation.
[0025] FIG. 1 illustrates the complete cDNA sequence of the
.alpha.(1,3) galactosyltransferase gene (SEQ. ID. No. 1), having an
open reading frame of 1113 base pairs, encoding a 371 amino acid
protein.
[0026] FIG. 2 compares the protein sequences encoded by the
porcine, bovine and murine .alpha.(1,3) galactosyltransferase cDNA
genes.
[0027] FIG. 3 illustrates the secondary structure of a transacting
hammerhead ribozyme targeted to .alpha.(1,3) galactosyltransferase
mRNA.
[0028] FIG. 4 illustrates the genomic organization of the murine
.alpha.(1,3-) galactosyltransferase gene. Exons are labeled 1-9 and
are indicated by solid boxes. Intron sequences are represented by a
thin line.
[0029] A method of producing a chimeric swine and porcine organs
and tissue cultures, homozygous for an .alpha.(1,3)
galactosyltransferase gene inactivation, in which .alpha.(1,3)
galactosyltransferase protein synthesis and cell surface
Gal.alpha.1-3Gal.beta.1-4GlcNAc epitope-containing carbohydrate
cell surface markers expression are deficient is disclosed. Of
particular interest are purified cell types which have been
rendered deficient in .alpha.(1,3) galactosyltransferase
expression. Such cell types include fibroblasts, keratinocytes,
myoblasts and endothelial cells.
[0030] In one embodiment of the present invention, swine cells
altered as described herein are used to provide cells needed by a
recipient or to provide gene therapy. The cells, which are
deficient in Gal.alpha.(1-3)Gal.beta.1-4GlcNAc epitope-containing
carbohydrates cell surface antigen, are cultured and transplanted
to an oocyte.
[0031] The embryonic stem cells with the null mutant .alpha.(1,3)
galactosyltransferase locus are introduced into swine blastocysts,
which are then introduced into a pseudopregnant swine. The embryo
develops into a chimeric swine offspring. When bred with wild-type
females, chimeric males transmit the .alpha.(1,3)
galactosyltransferase inactivation in the embryonic stem cell to
their offspring, which are heterozygous for the inactivation. Swine
heterozygous for the .alpha.(1,3) galactosyltransferase gene
inactivation can be intercrossed to produce swine homozygous (-/-)
for the mutation.
[0032] Purified or substantially pure .alpha.(1,3)
galactosyltransferase deficient cells can be obtained from tissues
or transgenic or chimeric swine produced as described herein.
Alternatively, they can be obtained from a normal (non-altered)
donor swine and altered using a method described herein. These
cells can be then cultured by known methods to produce a quantity
of cells useful for transplantation. In addition, cell lines, such
as swine cell lines, in which the .alpha.(1,3)
galactosyltransferase gene is disrupted, preferably on both
alleles, are useful as a source of tissue and cells for
transplantation.
EXAMPLE 1
Isolation and Characterization of Porcine .alpha.(1,3)
Galactosyltransferase cDNA
[0033] A previously described .lambda.ZAP II porcine spleen cDNA
library (Gustafsson et al. 1990. Proc. Natl. Acad. Sci. USA.
87:9798-9802) was screened by hybridization with a cloned
.alpha.(1,3) galactosyltransferase cDNA probe (Joziasse et al.
1989. J. Biol. Chem. 264:14290-14297) The Genbank Accession number
for the bovine .alpha.(1,3) galactosyltransferase cDNA sequence is
J04989. The bovine .alpha.(1,3) galactosyltransferase cDNA probe
was kindly provided by Dr. David Joziasse, University of Leiden,
The Netherlands. The probe was radioactively labeled with a
.sup.32P-dATP using the Megaprime DNA labeling system (Amersham
International, UK). Positive clones were confirmed by the
polymerase chain reaction (PCR), using primers (SEQ. ID. NO: 2 and
SEQ. ID. NO: 3) derived from the bovine .alpha.(1,3)
galactosyltransferase cDNA sequence. SEQ. ID. NO: 2 corresponds to
bovine .alpha.(1,3) galactosyltransferase nucleotides 712-729 and
SEQ. ID. NO: 3 corresponds to the reverse complement of bovine
.alpha.(1,3) galactosyltransferase nucleotides 1501-1508.
Recombinant pBluescript plasmids from positive clones were
automatically excised with the helper phage R408 (Stratagene Ltd.,
Cambridge, UK) and amplified in E. coli strain TG1 (ATCC 39078).
Plasmid DNA was prepared using the Magic Miniprep kit (Promega
Ltd., Southampton, UK) following the manufacturer's instructions
and the DNA was characterized by cleavage with EcoRI. DNA
sequencing was performed by the dideoxy chain termination method,
using a T7 DNA polymerase sequencing kit (Pharmacia Biosystems
Ltd., Milton Keynes, UK) according to the manufacturer's
instructions. The synthetic oligonucleotide primers SEQ. ID. NOs.
4-12 were used. SEQ. ID. NO. 4 is Stratagene SK, catalog number
300305, (Stratagene Inc., La Jolla, Calif.). SEQ. ID. NO: 5
corresponds to the reverse complement of porcine .alpha.(1,3)
galactosyltransferase nucleotides 94-111. SEQ. ID. NO: 6
corresponds to the porcine .alpha.(1,3) galactosyltransferase
nucleotides 163-180. SEQ. ID. NO: 7 corresponds to the reverse
complement of porcine .alpha.(1,3) galactosyltransferase
nucleotides 442-459. SEQ. ID. NO: 8 corresponds to the complement
of porcine and bovine .alpha.(1,3) galactosyltransferase
nucleotides 538-555 and 982-999, respectively. SEQ. ID. NO: 9
corresponds to the reverse complement of porcine .alpha.(1,3)
galactosyltransferase nucleotides 596-615. SEQ. ID. NO: 10
corresponds to the porcine .alpha.(1,3) galactosyltransferase
nucleotides 682-699. SEQ. ID. NO: 11 corresponds to the porcine
.alpha.(1,3) galactosyltransferase nucleotides 847-864. SEQ. ID.
NO: 12 corresponds to the reverse complement of porcine
.alpha.(1,3) galactosyltransferase nucleotides 970-987.
[0034] Four positive clones were obtained from approximately
2.times.10.sup.4 plaques screened by hybridization with the bovine
.alpha.(1,3) galactosyltransferase cDNA probe (Joziasse et al.
1989. J. Biol. Chem. 264:14290-14297). Three of these clones were
confirmed to be positive by PCR. Each of the three recombinant
pBluescript plasmids, generated by automatic excision from
.lambda.ZapII with helper phage, contained inserts of approximately
2.5 kb as determined by EcoRI cleavage. One clone, designated
pS.alpha.13GT1, was selected for further study.
[0035] DNA sequence analysis of pS.alpha.13GT1 revealed an open
reading frame of 1113 bases (See SEQ. ID. NO: 1 and FIG. 1) showing
86% identity with the published bovine cDNA sequence (Joziasse et
al. 1989. J. Biol. Chem. 264:14290-14297) and encoding a 371 amino
acid protein with 85% and 76% identity with the bovine and murine
.alpha.(1,3) galactosyltransferase amino acid sequences,
respectively (FIG. 2).
EXAMPLE 2
Antisense Oligonucleotide Inhibition of .alpha.( 1,3)
Galactosyltransferase Expression
[0036] Three antisense 5' and 3' phosphothioate-protected
oligonucleotides (S-oligonucleotides, SEQ. ID. NOs 13-15) are
tested in an in vitro system employing porcine primary endothelial
cell cultures (from porcine aorta) and a porcine B-cell line (L231,
European Collection of Animal Cell Cultures, PHLS, Center for
Applied Microbiology and Research, Porton Down, Salisbury, UK).
SEQ. ID. NO: 13 corresponds to the reverse complement of porcine
.alpha.(1,3) galactosyltransferase cDNA nucleotides 16-35. SEQ. ID.
NO: 14 corresponds to the reverse complement of porcine
.alpha.(1,3) galactosyltransferase cDNA nucleotides 31-53. SEQ. ID.
NO: 15 corresponds to the reverse complement of porcine
.alpha.(1,3) galactosyltransferase cDNA nucleotides 6-23. All three
antisense oligonucleotides are directed at the 5' region of the
mRNA surrounding the initiation of translation. Nonsense
S-oligonucleotides randomized from the antisense sequence are used
as controls at similar molar concentrations.
[0037] Porcine endothelial cells are derived from miniature swine
aorta by scraping the luminal surface of the blood vessel as
described (Ryan et al. Tissue and Cell 12:619-635). The cells are
suspended in M199 medium supplemented with 20% fetal bovine serum
(GIBCO BRL, Gaithersburg, Md.) and gentamycin and plated in 25
cm.sup.2 tissue culture flasks, pre-coated with fibronectin (5
.mu.g/cm.sup.2) and laminin (1 .mu.g/cm.sup.2). Endothelial cell
growth supplement (Collaborative Research, Bedford, Mass.) at 150
.mu.g/ml is added at the beginning of the culture. The cultures are
maintained by changing one half of the medium every 2-3 days. The
porcine lymphoblastoid cell line L231 is maintained in DMEM, 10%
fetal bovine serum, 10% NCTC-109, 1% glutamine, 1% pen-strep (all
from GIBCO BRL, Gaithersburg, Md.) and 5.times.10-.sup.2 M
2-mercaptoethanol (Sigma, St. Louis, Mo.). The L231 cells are
sub-cultured by splitting the cells 1:3 every three days.
[0038] For cell treatment, S-oligonucleotides are added, to a final
concentration of 5-10 .mu.M, to growing cells at 24 hr intervals,
typically for 48 hr, and then the treated cells are examined for
the levels of .alpha.(1,3) galactosyltransferase mRNA (by Northern
blot analysis) and expression of the epitope on the cell surface by
human AB serum and FITC-labeled mouse anti-human secondary reagents
(anti-human IgM and IgG).
EXAMPLE 3
Preparation and Use of Integrated Antisense Constructs
[0039] We are studying the ability of integrated antisense
constructs to inhibit specifically the production of the porcine
.alpha.(1,3) galactosyltransferase. Specific inhibition of the
.alpha.(1,3) galactosyltransferase in transfected cells allows
assessment of the contribution of the enzyme in the hyperacute
phenomenon. Vectors are constructed to express the .alpha.(1,3)
galactosyltransferase antisense mRNA, under the control of the
cytomegalovirus (CMV) promoter. Specifically, pS.alpha.13GT1 is
cleaved with NotI and EcoRV which generates a restriction fragment
of length 537 bp, containing part of the pBluescript polylinker
sequence through to nucleotide 531 of the .alpha.(1,3)
galactosyltransferase cDNA sequence. This DNA fragment is cloned
into the expression vector pcDNA3 (Invitrogen, San Diego, Calif.),
cleaved with the same enzymes. The resulting vector therefore
contains the porcine .alpha.(1,3) galactosyltransferase sequence in
the antisense direction, relative to the CMV promoter located in
pcDNA3. The construct is transfected, using electroporation or
other high efficiency methods for introducing DNA into mammalian
cells, into both porcine endothelial cells and the L231 porcine
lymphoblastoid cell lines (grown as described in Example 2). The
effect of the antisense RNA is monitored by both Northern blot
analysis of mRNA of the .alpha.(1,3) galactosyltransferase gene and
the degree of binding of human serum components (i.e., natural
antibodies).
EXAMPLE 4
Ribozyme Sequences That Inactivate Porcine .alpha.(1,3)
Galactosyltransferase mRNA
[0040] This example describes a method for construction of the
vectors which encode ribozyme sequences which are specifically
designed to cleave the porcine .alpha.(1,3) galactosyltransferase
mRNA sequence.
[0041] The design of the ribozyme sequences is based upon the
consensus cis-acting hammerhead ribozyme sequence (Ruffner et al.
1990. Biochemistry 29:10695-10702). We used the Zuker algorithm in
the suite of programs available from the Genetics Computer Group
(Madison, Wis.) to model the cis-acting hammerhead ribozyme
sequences (Denman, R. B. 1993. BioTechniques 15:1090-1095).
Ribozyme target sequences are identified within the .alpha.(1,3)
galactosyltransferase mRNA sequence. A ribozyme sequence file for
each potential ribozyme sequence is generated based on the target
sequence and using five nucleotides to connect the mRNA target
sequence with the catalytic strand of the ribozyme. The sequence
file is then folded into the lowest energy structure using RNAFOLD.
Sequences which have non-ribozyme structures are discarded. FIG. 3
illustrates one of the ribozyme-target RNA secondary structures,
using the ribozyme corresponding to SEQ. ID. NO. 16. The small
arrow indicates the cleavage site on the mRNA, between stem I and
stem III.
[0042] Synthetic oligonucleotides to encode the ribozymes (SEQ. ID.
NOs. 16-21) are made on an Applied BioSystems Oligonucleotide
synthesizer (Foster City, Calif.) with termini corresponding to the
overhangs of the restriction endonucleases NotI and XbaI. The
duplex DNA is cloned into the mammalian expression cloning vector
pcDNA3 (Invitrogen, Calif.). Expression of the ribozyme is under
the control of the CMV promoter present in pcDNA3. The transcripts
consist of approximately 140 nucleotides both 5' and 3' to the
ribozyme sequence. The expression level of the transcribed sequence
is ascertained by Northern blot analysis. If the RNA level is low
additional sequences will be included in the construct in order to
generate a longer and more stable transcript.
[0043] The construct is transfected using the electroporation
technique into porcine primary endothelial cells, porcine B cells
(L231). Also, the vector is co-transfected with plasmid expressing
the porcine .alpha.(1,3) galactosyltransferase into COS7 cells.
Since COS7 cells do not express an endogenous .alpha.(1,3)
galactosyltransferase, the effect of the presence of the ribozyme
on the expression of the introduced porcine .alpha.(1,3)
galactosyltransferase gene is easily ascertained.
EXAMPLE 5
Transgenic Swine Producing Antisense or Ribozyme RNA That Inhibit
.alpha.(1,3) Galactosyltransferase Synthesis
[0044] This approach requires direct microinjection of transgene
DNA into one of the pronuclei of a porcine embryo at the zygote
stage(one-cell embryo). Injected one-cell embryos are transferred
to recipient foster gilts for development to term (Hammer et al.
1985, Nature 315:680; Pursel et al. 1989, Science 244:1281)
[0045] Critical to successfully accomplishing this approach is the
age and weight of the donor pigs, preferentially the haplotype
specific mini-swine. Optimally, the animals are of age 8 to 10
months and weigh 70 to 85 lbs. This increases the probability of
obtaining adequate supply of one-cell embryos for microinjection of
the transgenes. In order to allow for accurate timing of the embryo
collections at that stage from a number of embryo donors, the gilts
are synchronized using a preparation of synthetic progesterone
(Regumate). Hormone implants are applied to designated gilts 30
days prior to the date of embryo collection. Twenty days later, ten
days prior to the date of collection, the implants are removed and
the animals are treated with additional hormones to induce
superovulation i.e. to increase the number of embryos for
microinjection. Three days following implant removal, the animals
are treated with 400 to 1000 IU of pregnant mare serum gonadotropin
(PMSG) and with 750 IU of human chorionic gonadotropin (hCG) three
to four days later. These animals are bred by artificial
insemination (AI) on two consecutive days following injection of
hCG.
[0046] Embryo collections are performed as follows: three days
following the initial injection of hCG, the animals are
anesthetized with an intramuscular injection of Telazol (3 mg/lb.),
Rompum (2 mg/lb.) and Atropine (1 mg/lb.). A midline laparotomy is
performed and the reproductive tract exteriorized. Collection of
the zygotes is performed by cannulating the ampulla of the oviduct
and flushing the oviduct with 10 to 15 ml phosphate buffered
saline, prewarmed to 39.degree. C. Following the collection, the
donor animals are prepared for recovery from surgery according to
USDA guidelines. Animals used twice for embryo collections are
euthanized according to USDA guidelines.
[0047] Injection of the transgene DNA into the pronuclei of the
zygotes is carried out as follows. Zygotes are maintained in HAM's
F-12 medium supplemented with 10% fetal calf serum at 38.degree. C.
in 5% CO.sub.2 atmosphere. For injection the zygotes are placed
into modified BMOC-2 medium containing HEPES salts (Ebert et al.
1984. J. Embryol. Exp. Morph. 84:91-103), centrifuged at
13,000.times.g to partition the embryonic lipids and visualize the
pronuclei. The embryos are placed in an injection chamber
(depression slide) containing the same medium overlaid with light
paraffin oil. Microinjection is performed on a Nikon Diaphot
inverted-microscope equipped with Nomarski optics and Narishige
micro-manipulators. Using 40.times. lens power the embryos are held
in place with a holding pipette and injected with a glass needle
which is back-filled with the solution of DNA containing the
transgene (2 .mu.g/ml). Injection of approximately 2 picoliters of
the solution (4 femptograms of DNA) which is equivalent to around
500 copies of the transgene is monitored by the swelling of the
pronucleus by about 50%. Embryos that are injected are placed into
the incubator prior to transfer to recipient animals.
[0048] Recipient animals are prepared similar to the donor animals,
but not superovulated. Prior to the transfer of the injected
embryos, recipient gilts are anesthetized, the abdomen opened
surgically by applying a longitudinal incision and the ovaries
exteriorized. The oviduct ipsilateral to the ovary with the larger
number of corpus lutei is flushed, the embryos checked to evaluate
if the animals is reproductively sound. Approximately 4 to 6
zygotes injected with the transgene are transferred to the flushed
oviduct, the abdominal incision sutured and the animals placed in a
warm area for recovery. The status of the pregnancy is monitored by
ultrasound starting at day 25, or approximately one week following
the expected date of implantation. Pregnant recipients are housed
separately until they are due to farrow.
[0049] Newborn piglets are analyzed for integration of the
transgene into chromosomal DNA. Genomic DNA is extracted from an
ear punch or a blood sample and initial screening is performed
using PCR. Animals that are potentially transgene-positive are
confirmed by Southern analysis. Transgenic founder animals are
subjected to further analysis including the levels of expression,
phenotype and germ line transmission. Northern analysis from RNA of
selective tissues including endothelial cells is performed to
determine the level of transgene expression. Also, immunological
assays including flow cytometric analysis for binding of antibody
from human serum and complement mediated lysis of pig cells
recognized by human natural antibodies are carried out to evaluate
the transgene effect. Animals that satisfy the above criteria are
used as founders for breeding of a transgenic line. If the founder
transgenic animals only satisfy part of the requirements, breeding
and specific intercrossing of transgenic offspring is performed to
assay the transgene effect in homozygous animals.
EXAMPLE 6
A Swine Made Null-mutant for .alpha.(1,3) Galactosyltransferase by
Homologous Recombination Using Porcine Embryonic Stem Cells
[0050] Gene targeting by homologous recombination in swine requires
several components, including the following: (A) a mutant gene
targeting construct including the positive/negative drug-selectable
marker genes (Tubulewicz et al. 1991. Cell 65:1153); (B) embryonic
stem cell cultures; and (C) the experimental embryology to
reconstitute an animal from the cultured cells.
[0051] The targeting construct is provided from a genomic clone
that spans most of the .alpha.(1,3) galactosyltransferase gene and
is isolated from a library made of isogenic DNA from a major
histocompatibility complex (MHC) haplotype d/d of the miniature
swine. Fragments of that genomic clone are introduced into a
positive/negative selectable marker cassette specifically developed
for gene targeting in embryonic stem (ES) cells and termed pPNT
(Tubulewicz et al. 1991. Cell 65:1153). This gene targeting
cassette includes as positive selectable marker the bacterial
neomycin phosphotransferase gene (neo) which allows for selection
of cells in G418. The neo gene is regulated by a promoter that
guarantees high level expression in ES cells such as the
phosphoglycerate kinase promoter-1 (PGK-1). Negative selection is
accomplished by expressing the Herpes Simplex Virus--thymidine
kinase (HSV-tk) gene which allows for selective killing of cells in
Gancyclovir. Similar to the neo gene, the HSV-tk gene is regulated
by the PGK-1 promoter, as well. In the targeting cassette pPNT
there are unique and convenient cloning sites between the neo and
the HSV-tk gene which are suitable sites to introduce the genomic
fragment of the .alpha.(1,3) galactosyltransferase gene upstream of
the translation initiation signal AUG (e.g. SalI sites in introns 2
and 4) This fragment of approximately 2 kb of DNA is cloned in
reverse orientation to the direction of transcription of the
PGK-neo cassette to assure that no truncated or residual peptide is
generated at the .alpha.(1,3) galactosyltransferase-locus. Genomic
sequences of the .alpha.(1,3) galactosyltransferase locus
downstream of exon 4, approximately 5 kb are introduced into pPNT
at the 5'-end of the neo gene. This targeting construction termed
pPNT-alpha GT1 is linearized and transfected by electroporation
into porcine ES cells. Double selection in G418 (150 to 300
.mu.g/ml) and Gancyclovir is performed to initially isolate clones
of ES cells with targeted mutations in the .alpha.(1,3)
galactosyltransferase locus. Confirmation of homologous recombinant
clones is achieved using Southern analysis.
[0052] ES cell clones that have undergone targeted mutagenesis of
one allele of the .alpha.(1,3) galactosyltransferase locus are
subjected to a second round of in vitro mutagenesis or used for
reconstituting an animal that contains the mutation. A second round
of in vitro mutagenesis can be carried out using an analogous
targeting construction with hygromycin phosphotransferase hyg as
positive selectable marker gene.
[0053] As far as the reconstitution of animals is concerned, the
methods include nuclear transfer, blastocyst injection or morula
aggregation. The preferred routes include either blastocyst
injection or morula aggregation which yield chimeras between the
donor cells and the recipient embryos. For both these methods
recipient embryos are prepared as follows: embryo donor/recipient
gilts are synchronized and mated as described in Example 5. On day
6 following artificial insemination or natural mating, the gilts
are prepared for surgery as described earlier, anesthetized and the
uteri retrogradely flushed using a prewarmed (38.degree. C.)
solution of phosphate buffered saline (PBS). Intact blastocysts
that are encapsulated by the zona pellucida are placed in a
depression slide containing HEPES-buffered medium (Whitten's or
TL-HEPES) and approximately 15 to 20 ES cells are injected into the
blastocoel using a glass injection needle with an opening of 20
.mu.m and Narishige micromanipulators. Injected embryos are then
reimplanted into recipient foster gilts for development to term and
pregnancies are monitored using ultrasound. Offspring is analyzed
for chimerism using the polymerase chain reaction (PCR) of DNA
samples extracted from blood, skin and tissue biopsies and primers
complementary to the neo or hyg gene. Germ line transmission of the
chimeras is assayed using PCR and in situ hybridization of tissue
samples obtain from male and female gonads. Male and female
chimeras which transmit the ES cell genotype to the germ line are
crossed to yield homozygously mutant animals. Analysis of mutant
animals for expression of .alpha.(1,3) galactosyltransferase and
binding of human natural antibodies to endothelial cells of those
animals is used as final test to assess the validity of gene knock
out approach in swine.
EXAMPLE 7
A Swine Made Null-mutant for .alpha.(1.3) Galactosyltransferase
Using Isogenic DNA Targeting into Zygotes
[0054] Isogenic DNA or DNA that is substantially identical in
sequence between the targeting vector and target DNA in the
chromosomes greatly increases the frequency for homologous
recombination events and thus the gene targeting efficiency. Using
isogenic DNA-targeting vectors, targeting frequencies of 80% or
higher are achieved in mouse embryonic stem cells. In contrast,
non-isogenic DNA-vectors normally yield targeting frequencies of
around 0.5% to 5%, i.e., approximately two orders of magnitude
lower than isogenic DNA vectors. Surprisingly, isogenic DNA
constructions are predominantly integrated into chromosomes by
homologous recombination rather than random integration. As a
consequence, targeted mutagenesis of genes can, therefore, be
carried out in biological systems, including animals and even
zygotes, which do not lend themselves to the use of elaborate
selection protocols.
[0055] The significance for isogenic DNA in gene targeting
approaches was first described by TeRiele et al., PNAS 89,
5128-5132 (1992) and also in PCT/US 92/07184.
[0056] In order for the isogenic DNA approach to be feasible,
targeting vectors have to be constructed from a source of DNA that
is identical to the DNA of the organism to be targeted. Ideally,
isogenic DNA targeting is carried out in inbred strains of animals
in which all genetic loci are homozygous. All animals of that
strain can therefore serve as a source for generating isogenic
targeting vectors. In cases in which inbred strains of animals are
lacking, genetic loci can consist of many different alleles. Thus,
isogenic vectors can only be derived from the individual in which
targeted gene replacement is attempted. Furthermore, the targeting
vector would be isogenic for one allele only.
[0057] In the haplotype specific miniature swine, isogenic gene
targeting vectors are readily derived for the MHC locus, since that
genomic region has been maintained homozygous for several
generations. Since the cDNA sequence to .alpha.(1,3)
galactosyltransferase locus has been determined (Example 1) and
genomic clones have been generated recently it is now possible to
map and thereby avoid, polymorphisms within the .alpha.(1,3)
galactosyltransferase locus gene.
[0058] Mapping of polymorphism is carried out as follows. A group
of mini-swine that are closely related, either siblings and/or
parent/offspring is identified. High molecular weight DNA is
extracted and PCR reactions are set up to amplify sequences from
Exon 6 to Exon 7 spanning Intron 6. The organization of Exons 5
through 9 of the porcine gene are known to be homologous to those
of the murine gene (FIG. 4, Joziasse, D. H., Shaper, N. L., Kim,
D., Van den Eijnden, D. and Joel H. Shaper, J. Biol. Chem. 167, pp.
5534-5541 (1992)). In FIG. 4, genomic organization of the murine
.alpha.(1,3-) galactosyltransferase gene Exons are labeled 1-9 and
are indicated by solid boxes. Intron sequences are represented by a
thin line. The 5'-oligo in Exon 6 has the following sequence:
5'-TCC GAG CTG GTT TAA CAA TGG GTA-3' (SEQ ID NO: 25) and the 3'
oligo in Exon 7 contains the following sequence: 5'-TCT TCG TGG TAA
CTG TGA GTC CTA-3' (SEQ ID NO: 26). The PCR fragments are
approximately 1 and 2 kb long. In order to assay for PCR-RFLPs,
restriction digestions using 4-cutters are carried out which are
expected to cut frequently. Such 4-cutters include BstU I (CGCG),
Rae III (GGCC), Hha I (GCGC) Sau3A (GATC), Rsa I (GTAC) and Taq I
(TCGA), which are commercially available from New England BioLabs
(Beverley, Mass.). Based on the pattern of restriction fragments
obtained from parental animals and offspring, as well as between
siblings, it is possible to define the number of different alleles
present in that group of animals. In case the PCR-RFLP does not
yield obvious sequence polymorphisms between different alleles in
different animals, alternative methods can be employed including
DNA sequencing or enzyme mismatch cleavage (EMC) using T7 or T4
resolvases. Additional animals are then analyzed in a similar
manner to identify specific alleles at the .alpha. 1,3
galactosyltransferase locus. Once several alleles have been
identified, targeting constructions are prepared for those alleles
that are most abundantly present in the line of mini-swine selected
for zygote injection.
[0059] Gene targeting constructions are generated as follows. A
genomic library is made in lambda replacement vectors including
Lambda FIX II, Lambda EMBL4 or Lambda Dash II (Stratagene Cloning
Systems). The library is then plated at a density of around 50,000
plaques/15 cm plate and screened with a probe specific for Exon 9
of the swine .alpha.(1,3) galactosyltransferase gene. The probe is
generated using PCR which is carried out using a standard protocol:
35 cycles, anneal at 60.degree. C. for 20 sec, extend at 72.degree.
C. for 40 sec and denature at 94.degree. C. for 20 sec. Positive
clones with insert sizes of around 15 kb are then subcloned into
plasmid vectors including pSP72 and engineered for targeting as
either an insertion vector or a replacement vector.
[0060] An insertion vector is designed as described by Hasty and
Bradley (Gene Targeting Vectors for Mammalian Cells, in Gene
Targeting: A Practical Approach, ed, Alexandra L. Joyner, IRL Press
1993). Insertion vectors require for only one crossover to occur
for integration by homologous recombination into the native locus.
The double strand breaks, the two ends of the vector which are
known to be highly recombinogenic, are located on adjacent
sequences on the chromosome. Preferably the site of integration of
the insertion vector is at the beginning of Intron 8 of the
.alpha.(1,3) galactosyltransferase gene. The entire length of
Intron 8 is thereby included as a homologous and identical
sequence, carrying the minimal number mismatches. Exon 9 which
encodes the catalytic domain of the enzyme is then substituted by a
selectable marker such as the neomycin phosphotransferase gene, or
a derivative thereof termed IRES .beta.geo. Downstream, or 3' of
the selectable marker, sequences from Intron 7, Exon 8 and Intron 8
are included, terminating at the site adjacent to the beginning of
the vector. The targeting frequencies of that construction are in
the range of 30 to 50%. All these constructions are made using
standard cloning procedures.
[0061] The replacement vectors have also been extensively described
by Hasty and Bradley, supra. Conceptually more straightforward than
the insertion vector, replacement vectors issued here are
essentially co-linear fragments of a certain stretch of genomic
sequence at the .alpha.(1,3) galactosyltransferase locus.
Preferably, the DNA sequence from which an isogenic replacement
vector is constructed include approximately 6 to 10 kb of
uninterrupted DNA. This DNA corresponds to a region between the
start of Exon 4 and the end of Intron 8 of certain alleles of the
.alpha.(1,3) galactosyltransferase locus from mini-swine. Exon 9,
encoding the catalytic domain is replaced with the selectable
marker gene, such as the neo gene, or a functional analog thereof.
The 3'-end of the replacement vector includes the 3' untranslated
region of the gene in Exon 9, including approximately 1 kb of
isogenic DNA. Two crossovers, one on either side of the selectable
marker causes the mutant targeting vector to become integrated and
replace the wild-type gene.
[0062] Microinjection of the isogenic transgene DNA into one of the
pronuclei of a porcine embryo at the zygote stage (one-cell
embryo), particularly the mini-swine, is accomplished by
modification of the protocol described earlier. (Hammer et al.
1985, Nature 315, 680; Pursel et al 1989, Science 244, 1281).
Optimally, the mini-swine are of age 8 to 10 months and weigh 70 to
85 lbs. This increases the probability of obtaining an adequate
supply of one-cell embryo donors. The gilts are synchronized using
a preparation of synthetic progesterone (Regumate). Hormone
implants are applied to designated gilts 30 days prior to the date
of embryo collection. Twenty days later, ten days prior to the date
of collection, the implants are removed and the animals are treated
with additional hormones to induce superovulation, i.e., to
increase the number of embryos for microinjection. Three days
following implant removal, the animals are treated with 400 to 1000
IU of pregnant mare serum gonadotropin (PMSG) and with 750 IU of
human chorionic gonadotropin (hCG) three to four days later. These
animals are bred by artificial insemination (AI) on two consecutive
days following injection of hCG.
[0063] Embryo collections are performed as follows. Three days
following the initial injection of hCG, the animals are
anesthetized with an intramuscular injection of Telazol (3 mg/lb),
Rompum (2 mg/lb) and Atropine (1 mg/lb). A midline laparotomy is
performed and the reproductive tract exteriorized. Collection of
the zygotes is performed by cannulating the ampulla of the oviduct
and flushing the oviduct with 10 to 15 ml phosphate buffered
saline, prewarmed to 39.degree. C. Following the collection, the
donor animals are prepared for recovery from surgery according to
USDA guidelines. Animals used twice for embryo collections are
euthanized according to USDA guidelines.
[0064] Injection of the transgene DNA into the pronuclei of the
zygotes is carried out as summarized below. Zygotes are maintained
in HAM F-12 medium supplemented with 10% fetal calf serum at
38.degree. C. in 5% CO.sub.2 atmosphere. For injection the zygotes
are placed into BMOC-2 medium, centrifuged at 13,000 g to partition
the embryonic lipids and visualize the pronuclei. The embryos are
placed in an injection chamber (depression slide) containing the
same medium overlaid with light paraffin oil. Microinjection is
performed on a Nikon Diaphot inverted-microscope equipped with
Nomarski optics and Narishige micromanipulators. Using 40.times.
lens power the embryos are held in place with a holding pipette and
injected with a glass needle which is back-filled with the solution
of DNA containing the transgene (2 .mu.g/ml). Injection of
approximately 2 picoliters of the solution (4 femptograms of DNA)
which is equivalent to around 500 copies of the transgene is
monitored by the swelling of the pronucleus by about 50%. Embryos
that are injected are placed into the incubator prior to transfer
to recipient animals.
[0065] Recipient animals are prepared similar to the donor animals,
but not superovulated. Prior to the transfer of the injected
embryos, recipient gilts are anesthetized, the abdomen opened
surgically by applying a longitudinal incision and the ovaries
exteriorized. The oviduct ipsilateral to the ovary with the larger
number of corpus lutei is flushed and the embryos are checked to
evaluate if the animal is reproductively sound. Approximately 4 to
6 zygotes injected with the transgene are transferred to the
flushed oviduct, the abdominal incision sutured and the animals
placed in a warm area for recovery. The status of the pregnancy is
monitored by ultrasound starting at day 25, or approximately one
week following the expected date of implantation. Pregnant
recipients are housed separately until they are due to farrow.
[0066] Newborn piglets are analyzed for integration of the
transgene into chromosomal DNA. Genomic DNA is extracted from an
ear punch or a blood sample and initial screening is performed
using PCR. Animals that are potentially transgene-positive are
confirmed by Southern analysis. Transgenic founder animals are
subjected to further analysis regarding the locus of transgene
integration using Southern analysis. Those animals that have the
transgene targeted to the native .alpha.(1,3) galactosyltransferase
locus, i.e., that are heterozygous mutant for that gene, are grown
up and bred to additional heterozygous mutant founder animals.
Applying Mendelian genetics allows us to breed animals that are
homozygous mutant for the .alpha.(1,3) galactosyltransferase.
Functional assays using the lectin BI4 and human preformed natural
antibodies are then used to look at expression of the Gal
.alpha.(1,3) Ga1 epitope on swine cells and to confirm the knockout
of the .alpha.(1,3) galactosyltransferase gene.
Sequence CWU 1
1
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