U.S. patent application number 14/032934 was filed with the patent office on 2014-06-26 for method of producing an antibody.
This patent application is currently assigned to Central Institute for Experimental Animals. The applicant listed for this patent is Central Institute for Experimental Animals, Hisako Nomura. Invention is credited to Kiyoshi Ando, Sonoko Habu, Mamoru Ito, Kimio Kobayashi, Yoshio Koyanagi, Tatsutoshi Nakahata, Tatsuji Nomura, Kazuo Sugamura, Koichiro Tsuji, Naoki Yamamoto.
Application Number | 20140178408 14/032934 |
Document ID | / |
Family ID | 18837744 |
Filed Date | 2014-06-26 |
United States Patent
Application |
20140178408 |
Kind Code |
A1 |
Ito; Mamoru ; et
al. |
June 26, 2014 |
METHOD OF PRODUCING AN ANTIBODY
Abstract
The present invention provides an immunodeficient mouse (NOG
mouse) suitable for engraftment, differentiation and proliferation
of heterologous cells, and a method of producing such a mouse. This
mouse is obtained by backcrossing a C.B-17-scid mouse with an
NOD/Shi mouse, and further backcrossing an interleukin 2-receptor
.gamma.-chain gene-knockout mouse with the thus backcrossed mouse.
It is usable for producing a human antibody and establishing a stem
cell assay system, a tumor model and a virus-infection model.
Inventors: |
Ito; Mamoru; (Kanagawa,
JP) ; Kobayashi; Kimio; (Kanagawa, JP) ;
Nakahata; Tatsutoshi; (Kyoto, JP) ; Tsuji;
Koichiro; (Tokyo, JP) ; Habu; Sonoko; (Tokyo,
JP) ; Koyanagi; Yoshio; (Chiba, JP) ;
Yamamoto; Naoki; (Tokyo, JP) ; Sugamura; Kazuo;
(Miyagi, JP) ; Ando; Kiyoshi; (Kanagawa, JP)
; Nomura; Tatsuji; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Nomura; Hisako
Central Institute for Experimental Animals |
Shibuya-Ku
Kanagawa |
|
JP
JP |
|
|
Assignee: |
Central Institute for Experimental
Animals
Kanagawa
JP
|
Family ID: |
18837744 |
Appl. No.: |
14/032934 |
Filed: |
September 20, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12620469 |
Nov 17, 2009 |
8541033 |
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14032934 |
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11517698 |
Sep 8, 2006 |
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12620469 |
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10221549 |
Sep 10, 2002 |
7145055 |
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PCT/JP01/09401 |
Oct 25, 2001 |
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11517698 |
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Current U.S.
Class: |
424/172.1 ;
435/70.4 |
Current CPC
Class: |
A01K 67/027 20130101;
A01K 2267/0331 20130101; A01K 2267/0337 20130101; A01K 2267/03
20130101; A61K 39/00 20130101; C07K 16/18 20130101; A01K 67/0271
20130101 |
Class at
Publication: |
424/172.1 ;
435/70.4 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Claims
1-11. (canceled)
12. A method of producing a human antibody comprising: introducing
human B-cells, or both human B cells and T-cells, or human stem
cells which differentiate into human B-cells or both human B cells
and T-cells, into a mouse produced by a method comprising
backcrossing a mouse B with a mouse A, wherein said mouse A is a
mouse obtained by backcrossing a Severe Combined Immunodeficiency
(SCID) mouse with a non-obese diabetic (NOD) mouse; and wherein
said mouse B is an interleukin 2-receptor .gamma. chain gene
knockout mouse, wherein said mouse produced by the method retains
the human B-cells or both human B cells and T-cells; immunizing
said mouse with an antigen; and collecting the human antibody
produced from the mouse.
13. A method of producing an antibody-producing cell line which
produces a human antibody, comprising: introducing human B-cells,
or both human B cells and T-cells, or human stem cells which
differentiate into human B-cells or both human B cells and T-cells,
into a mouse produced by a method comprising backcrossing a mouse B
with a mouse A, wherein said mouse A is a mouse obtained by
backcrossing a Severe Combined Immunodeficiency (SCID) mouse with a
non-obese diabetic (NOD) mouse; and wherein said mouse B is an
interleukin 2-receptor .gamma. chain gene knockout mouse, wherein
said mouse produced by the method retains the human B-cells or both
human B cells and T-cells; immunizing said mouse with an antigen;
collecting from the mouse a cell which produced the antibody
against the antigen; and establishing a cell line of the cell.
14. The method of claim 12, wherein the human B-cells, or both
human B cells and T-cells, or human stem cells which differentiate
into human B-cells or both human B cells and T-cells, are
introduced intravenously.
15. The method of claim 12, wherein the human stem cells which
differentiate into human B-cells or both human B cells and T-cells,
are CD34+ cells.
16. The method of claim 13, wherein the antibody-producing cell is
collected from the spleen.
17. The method of claim 13, wherein the antibody-producing cell is
collected from a lymph node.
18. The method of claim 12, wherein the human stem cells which
differentiate into human B-cells or both human B cells and T-cells
are cord blood cells.
19. The method of claim 13, wherein the human stem cells which
differentiate into human B-cells or both human B cells and T-cells
are cord blood cells.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/620,469, filed Nov. 17, 2009, which will issue as U.S. Pat.
No. 8,541,033 on Sep. 24, 2013, which claims priority under 35 USC
.sctn.120 to U.S. application Ser. No. 11/517,698 filed Sep. 8,
2006, now abandoned, which is a divisional of, and claims priority
under 35 USC .sctn.120 to, U.S. application Ser. No. 10/221,549
filed Sep. 10, 2002, now U.S. Pat. No. 7,145,055 issued Dec. 5,
2006, which is a .sctn.371 filing of PCT Application
PCT/JP01/09401, filed Oct. 25, 2001, the entire disclosures of
which are hereby incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates to a method of producing an
excellent mouse for engraftment of heterologous cells, a mouse
produced by this method and use of the mouse.
BACKGROUND ART
[0003] Laboratory animals to which heterologous cells including
human cells are engrafted are very important for analysis of onset
mechanisms of various diseases and drug developments for the
treatments or preventions thereof, and development of animals as
receptors therefor is one of major themes in laboratory animal
sciences. In particular, in recent years, treatments etc. (known as
regenerative medicine) in which tissues or cells differentiated
from stem cells are transplanted have received world-wide
attention, and therefore these animals are of increasing
importance.
[0004] The inventors of the present invention have continued to
develop and improve these laboratory animals. In particular, they
made improvements or the like on a nude mouse or a SCID mouse, and
they have already filed a patent application (Japanese Patent
Application Laying-Open (kokai) No. 9-94040) concerning an
immunodeficient mouse etc. produced for this purpose. Above all, an
NOD/Shi-scid mouse and an NOD/LtSz-scid mouse which exhibit
multifunctional immunodeficiency (functional deficiency of T cells
and B cells, decline of macrophage function, reduction of
complement activity, reduction of natural killer (NK) activity
etc.) are the most noteworthy as laboratory animals suitable for
engraftment of heterologous cells. Since it became clear that they
could be used for various types of research including stem cell
differentiation and proliferation, the range of applications in
which they are used has increased to the present level.
[0005] However, human cells are engrafted to the NOD/Shi-scid mouse
at a high ratio, but it is recognized that the engraftment capacity
is substantially varied.
[0006] In order to enhance the engraftment capacity of the
NOD/Shi-scid mouse, it has already been revealed that reduction of
NK activity in the mouse by administering anti IL-2R.beta. chain
antibodies (TM.beta.1), anti-asialo-GM1 antibodies or the like is
important. (Koyanagi, Y. et al., 1997. "Primary human
immunodeficiency virus type 1 viremia and central nervous system
invasion in a novel hu-PBL-immunodeficient mouse strain." J Virol
71:2417; Koyanagi, Y. et al., 1997. "High levels of viremia in
hu-PBL-NOD-scid mice with HIV-1 infection." Leukemia 11 Suppl.
3:109; Yoshino H, et al., 2000. "Natural killer cell depletion by
anti-asialo GM1 antiserum treatment enhances human hematopoietic
stem cell engraftment in NOD/Shi-scid mice." Bone Marrow Transplant
26:1211-6. However, these antibodies are very expensive, and it is
recognized that their efficacies vary between individuals. Further,
when anti-asialo GM1 antibodies are used, the administration
thereof should be conducted with the frequency of every eleventh
day during the experiment period, and thus a degree of complexity
is attached.
[0007] Therefore, Dr. Shultz, L. D. et al. of The Jackson
Laboratory in the United States produced an NOD/LtSz-scid, .beta.2m
null (.beta.2m (null) NOD/SCID) mouse (Kollet O, Peled A, Byk T et
al., beta2 microglobulin-deficient (.beta.2m(null))NOD/SCID mice
are excellent recipients for studying human stem cell function.
Blood 2000; 95(10):3102-5) by crossing an NOD/LtSz-scid mouse
having high engraftment capacity of human cells with a .beta.2m KO
mouse from which NK activity has been depleted.
[0008] With respect to the NOD/LtSz-scid, .beta.2m null mouse, T
cells, B cells and natural killer (NK) cells are depleted, and the
function of macrophages and complements is reduced. However, other
cells (e.g. dendritic cells) and factors (e.g. IFN.gamma.) are also
involved in the rejection of transplanted heterologous cells or
tissues.
[0009] Accordingly, a mouse which, compared to the NOD/LtSz-scid,
.beta.2m null mouse, has no variation in heterologous cell
engraftment capacity, requires no antibodies, and has excellent
heterologous cell engraftment is desirable.
DISCLOSURE OF THE INVENTION
[0010] Thus, the object of the present invention is to solve the
above problems and to provide a method of producing a mouse having
excellent heterologous cell engraftment capacity and a mouse
produced by the same method.
[0011] The present inventors have made intensive studies to solve
the above problems. As a result, they have obtained the findings
that a mouse which has no variation in engraftment capacity of
heterologous cells and requires no antibodies (that is, is suitable
for the engraftment of heterologous cells) can be obtained by
backcrossing an NOD/Shi mouse with a C.B-17-scid mouse, and further
backcrossing the thus obtained mouse with an interleukin 2-receptor
.gamma.-chain (IL-2R.gamma.) gene-knockout mouse. The present
invention has been accomplished based on the above findings.
[0012] Namely, the present invention is as follows.
[0013] (1) A method of producing a mouse suitable for engraftment
of heterologous cells, comprising backcrossing a mouse B with a
mouse A, as described below:
[0014] A: a mouse obtained by backcrossing a C.B-17-scid mouse with
an NOD/Shi mouse; and
[0015] B: an interleukin 2-receptor .gamma.-chain (IL-2R.gamma.)
gene knockout mouse.
[0016] (2) The method of producing a mouse as described in (1),
wherein the mouse A is an NOD/Shi-scid mouse.
[0017] 3) The method of producing a mouse as described in (1) or
(2), wherein the mouse B is an IL-2R.gamma.KO mouse.
[0018] (4) A mouse produced by the method of producing a mouse
described in any of (1) to (3).
[0019] (5) A NOG (NOD/Shi-scid, IL-2R.gamma. KO) mouse having
excellent engraftment capacity of heterologous cells, wherein both
of functional T-cells and functional B-cells are deleted,
macrophage function is reduced, NK cells or NK activity are
eliminated, dendritic cell function is reduced.
[0020] (6) The NOG mouse described in (4) or (5), wherein
transplanted human stem cells efficiently differentiate and
proliferate without being eliminated.
[0021] (7) A stem cell assay method comprising transplanting human
stem cells to the mouse described in any of (4) to (6) and
analyzing the differentiated and proliferated cells.
[0022] (8) The stem cell assay method described in (7), comprising
analyzing the differentiation and proliferation of T-cells and
B-cells.
[0023] (9) A method of proliferating human stem cells comprising:
[0024] transplanting and proliferating the human stem cells to the
mouse described in any of (4) to (6); [0025] collecting the human
stem cells from bone marrow of the mouse; and [0026] repeatedly
transplanting the collected cells to the mouse described in any of
(4) to (6).
[0027] (10) The method of proliferating human stem cells described
in (9), wherein the frequency of repeating is at least three
times.
[0028] (11) Human stem cells obtained by the method of (9) or (10),
wherein the obtained human stem cells have a purity of 99.7% or
more.
[0029] (12) The method described in (9) or (10), wherein the human
stem cells have foreign genes introduced thereinto.
[0030] (13) The mouse described in any of (4) to (6), wherein the
mouse is capable of stably retaining human T-cells and B cells and
producing a human antibody.
[0031] (14) A method of producing a human antibody comprising
immunizing with an antigen the mouse described in any of (4) to (6)
which retains human T-cells and B-cells.
[0032] (15) A method of producing an antibody-producing cell line
which produces a human antibody, comprising: [0033] immunizing with
an antigen the mouse described in any of (4) to (6) which retains
human T-cells and B-cells; [0034] collecting from the mouse cells
which produce the ntibody against the antigen; and [0035]
establishing a cell line.
[0036] (16) A human tumor model mouse wherein the mouse is a mouse
described in any of (4) to (6) and retains human tumor cells.
[0037] (17) The human tumor model mouse described in (16), wherein
the human tumor cells are derived from HTLV-1 leukemia.
[0038] (18) The human tumor model mouse described in (16) or (17),
wherein the mouse has the human tumor cells at an auricle
thereof.
[0039] (19) A method of screening an anticancer agent using the
mouse described in any of (16) to (18).
[0040] (20) A method of producing a human tumor model mouse
comprising transplanting human tumor cells to the mouse described
in any of (4) to (6).
[0041] (21) The method described in (16), wherein the human tumor
cells are derived from HTLV-1 leukemia.
[0042] (22) The method described in (20) or (21), wherein the human
tumor cells are transplanted at an auricle of the mouse.
[0043] (23) A virus-infected model mouse wherein the mouse is a
mouse described in any of (4) to (6) and retains T cells infected
with a T-tropic (T-cell affinity) virus as well as a
macrophage-tropic virus.
[0044] (24) The virus-infected model mouse described in (23),
wherein the virus is HIV.
[0045] (25) The virus-infected model mouse described in (23),
wherein the virus is HTLV-1.
[0046] (26) A method of screening an antiviral agent wherein the
method is carried out using the mouse described in (23) to
(25).
[0047] (27) A method of producing an immunodeficient mouse which
has engraftment capacity of enhanced heterologous cells compared
with a NOG mouse, wherein the method is carried out using the mouse
described in (3) to (6).
[0048] (28) The mouse described in (3) to (6), wherein the mouse is
used for producing an immunodeficient mouse which has enhanced
engraftment capacity of heterologous cells compared with a NOG
mouse.
[0049] Hereinafter, general embodiments of the present invention
will be described.
1. Production of a Mouse of the Present Invention
[0050] According to the present invention, a method of producing a
mouse suitable for the engraftment of heterologous cells is
characterized in backcrossing a mouse B with a mouse A, as
described below.
[0051] A: a mouse obtained by backcrossing a C.B-17-scid mouse with
an NOD/Shi mouse; and
[0052] B: an interleukin 2-receptor .gamma.-chain gene knockout
mouse.
[0053] Here, examples of the heterologous cells include cells or
tissues derived from mammals such as humans, mice, rats etc.,
particularly human stem cells, lymphocytes or tumor cells etc. of
humans, but not limited thereto.
[0054] With respect to the mouse A, backcrossing the C.B-17-scid
mouse with the NOD/Shi mouse is done in accordance with methods
well-known to a person skilled in the art, for example,
backcrossing by Cross Intercross method (Inbred Strains in
Biomedical Research, M. F. W. Festing, 1979, ISBN 0-333-23809-5,
The Macmillan Press, London and Basingstoke). The C.B-17-scid mouse
is crossed with the NOD/Shi mouse, and the obtained F1 mice are
further crossed with each other. Then, the immunoglobulin amount in
blood serum of the thus obtained F2 mice is measured for selecting
a mouse, from which immunoglobulin cannot be detected. The selected
mouse is again crossed with a NOD/Shi mouse. Repeating this process
(Cross Intercross method) 9 times or more enables the
accomplishment of the backcrossing.
[0055] A NOD/Shi mouse and a C.B-17-scid mouse are both
commercially available from CLEA JAPAN, INC. Further, examples of
mice obtained by crossing these mice with each other include a
NOD/Shi-scid mouse (also called as a NOD-scid mouse) (Japanese
Patent Application Laying-Open (kokai) No. 9-94040) which the
present inventors have already established. This mouse is purchased
from CLEA JAPAN, INC., and can be used directly as the mouse A. In
addition, the present inventors possess, other than the ones
mentioned above, NOD/Shi mice and NOD/Shi-scid mice, which can be
split up and provided whenever the need arises.
[0056] Moreover, with respect to the mouse B, knockout of an
interleukin 2-receptor .gamma.-chain (IL-2R.gamma.) gene is carried
out in accordance with methods well known to a person skilled in
the art, for example, a homologous recombination method using mouse
ES cells (Capecchi, M. R., Altering the genome by homologous
recombination, Science, (1989) 244, 1288-1292). After substituting
a specific mouse-derived gene by a homologous gene including a gene
resistant to a drug, for example neomycin etc. at ES cell stage,
the ES cells are inserted into a fertilized egg, thereby
accomplishing the gene-knockout.
[0057] Specifically, for example, gene clones containing a mouse
IL-2R.gamma. are isolated, from a genome library of 129/SV mouse,
using a human IL-2R.gamma.cDNA as a probe. Using a fragment of 8.6
kb containing the full length of IL-2R.gamma. among the clones, a
targeting vector is prepared. That is, PMCl-neo poly A which
expresses a neomycin resistant gene, is inserted between exons 7
and 8 of IL-2R in the fragment, and also a diphtheria toxin-A gene
is placed at 3' side 1 kb away from exon 8. Next, the vector is
made linear, and introduced into 1.times.10.sup.7 of E14 ES cells
by electroporation. Thereafter, ES clones which bring about
homologous recombination in the culture solution including G418,
are selected (confirmed by PCR or Southern method), and after
injecting the ES clones into blastocysts of C57BL/6 mice, they are
transplanted into the uteruses of foster parent mice. Chimeric mice
born from the foster parent mice are further crossed with C57BL/6
mice, thereby obtaining IL-2R.gamma.KO hetero mice wherein knockout
is transduced to germ cells.
[0058] Alternatively, pre-established interleukin-2 receptor
.gamma. chain gene (IL-2R.gamma.) knockout mouse strain may
directly be obtained for use from suppliers, and examples of the
mouse strains include interleukin-2 receptor .gamma. chain
(IL-2R.gamma.) knockout mice (Ohbo K, Suda T, Hashiyama M et al.,
Modulation of hematopoiesis in mice with a truncated mutant of the
interleukin-2 receptor gamma chain. Blood 1996; 87(3):956-67))
which was produced from IL-2R.gamma.KO mouse strains [Prof. Kazuo
Sugamura, Department of Microbiology and Immunology, Tohoku
University School of Medicine]. Incidentally, IL-2R.gamma.KO mice
are presently stored in the embryo preservation bank of the
applicants (Central Institute for Experimental Animals) at the
request of Prof. Sugamura, a producer of the mouse strain, and
whenever the need arises they can be provided as frozen embryos or
as thaw-reconstruction mice.
[0059] Further, backcrossing the mouse B with the mouse A can be
carried out, in similar fashion as described above, according to
conventional methods well-known to a person skilled in the art. For
example, in accordance with the above backcrossing, that is, a
NOD/Shi-scid mouse is crossed with an IL-2R.gamma.KO mouse, and the
obtained F1 mouse is backcrossed with an NOD/Shi-scid mouse,
thereby accomplishing the backcross.
[0060] Furthermore, a mouse of the present invention is
characterized in that the mouse is produced by the above method of
the present invention. The mouse of the present invention is
referred to as a NOG mouse (NOG mouse; NOD/Shi-scid, .gamma.c null
mouse; NOD/Shi-scid, IL-2R.gamma. chain-/-mouse; NOD/Shi-scid,
IL-2R(.gamma.c).sup.null mouse etc.)
[0061] The mouse of the present invention is a severe
immunodeficient mouse in which has both of functional T-cells and
functional B-cells are deleted, macrophage function are reduced,
and NK cells or NK activity are eliminated. Therefore, when
heterologous cells (e.g. human peripheral blood mononuclear
leukocytes) are introduced into the mouse of the present invention,
much higher ratios of engraftment and proliferation are observed
even in comparison with conventional immunodeficient mice which are
subjected to anti NK antibody treatment. (See Example 1 described
below.) Further, dendritic cells of the mouse of the present
invention are also functionally incompetent, and the production of
cytokine is remarkably reduced. Thus, the mouse of the present
invention has the most excellent engraftment capacity of
heterologous cells as compared with conventional immunodeficient
mice, and it is considered effective for analyses of various
introduced heterologous cells (including stem cells, differentiated
cells and cancer cells) which are engrafted in this mouse.
Additionally, it is possible to use the mouse in order to establish
a pathologic model mouse and produce a human antibody for HIV,
HTLV-1 or cancer.
[0062] Hereinafter, the applications of the mouse of the present
invention will be described. Usually, the mouse to be used is
preferably 8 to 12 weeks old, but not limited thereto.
2. Establishment of Human Stem Tell Assay System Using the Mouse of
the Present Invention
[0063] Using the mouse of the present invention, it is possible to
establish a human stem cell assay system for examining factors and
mechanisms which are engaged in differentiation and proliferation
of human stem cells. Also, it is possible to research various
therapeutic products using the human stem cell assay system.
[0064] Introducing human stem cells into the mouse of the present
invention enables the establishment of the human stem cell assay
system. Here, stem cells include, in addition to hematopoietic stem
cells, stem cells not derived from the hematopoietic system, such
as neural stem cells etc. Human stem cells are identified by the
existence of a cell surface marker which relates to a specific
epitope site identified by an antibody, and for example, they can
be isolated as CD34 positive cells from e.g. human bone marrow,
umbilical cord blood, peripheral blood etc.
[0065] Stem cells are suspended in a solution such as physiological
saline, phosphate buffered physiological saline etc., which exerts
no influence on cells and living organisms, and 1.times.10.sup.4 to
1.times.10.sup.6 of cells are intravenously administered into the
mouse, thereby carrying out the transplantation.
[0066] Cells are collected, several weeks after transplanting, from
each organ such as peripheral blood, the spleen, bone marrow, the
thymus etc. of the mouse to which the cells have been transplanted.
The surface antigens of these cells are examined using e.g. FACS
(Fluorescence-activated cell sorter), and thereby the
differentiation of the transplanted cells is examined. In this
case, examples of cell surface antigen markers to be used as index
include: CD34 which relates to stem cells; CD3, CD4, CD8 etc. which
relate to T-cells; CD10, CD19, CD20 etc. which relate to B-cells;
CD5 etc. which relate to B1a cells; CD33 etc. which relate to
myeloid cells; CD11c etc. which relate to dendritic cells; CD45
etc. which relate to the whole leukocytes; CD11a, CD11b etc. which
relate to macrophages; CD56 etc. which relate to NK cells; CD38
etc. which relate to plasma cells; CD41 etc. which relate to
platelets; and glycophorin A etc. which relate to erythrocytes.
According to need, various related markers can be selected.
[0067] The production of cytokines such as interferon, interleukin,
TNF.alpha. etc. in the collected cells, is measured by ELISA etc.,
and thereby the differentiation of the stem cells is examined.
[0068] Further, it is possible to conduct successive
transplantations of human stem cells using the mouse of the present
invention. That is, true self-replicable human stem cells can be
obtained. Specifically, human stem cells are transplanted in the
mouse of the present invention, after several weeks
undifferentiated human stem cells are collected from bone marrow of
the mouse, and further the collected stem cells are transplanted in
the mouse of the present invention. By repeating the
transplantation and collection, human stem cells which are free
from other cells and have high purity can be obtained in large
quantities. By the successive transplantations, human stem cells
with at least 99% or more purity, preferably 99.7% or more purity
can be obtained. Conventional mice allow up to secondary
transplantation, though the mouse of the present invention enables
more than two successive transplantations.
[0069] For the treatment of leukemia or the like, human stem cells
obtained using the mouse of the present invention can be
transplanted to humans. Also, the mouse is usable for gene therapy
targeting human stem cells by introducing a foreign gene into human
stem cells and transplanting them to the mouse of the present
invention for proliferation. With the aid of virus vectors such as
lentivirus vectors, retrovirus vectors, adenovirus vector, and
adeno-associated virus vector, a gene can be introduced into stem
cells. Examples of the genes to be used here include an ADA gene
for adenosine deaminase deficiency (ADA) patients. After these
genes are introduced into human stem cells, the cells are
transplanted to the mouse of the present invention for
proliferation and purification and then administered to patients,
thereby enabling gene therapy.
3. Production of Human Antibodies Using the Mouse of the Present
Invention
[0070] Using the mouse of the present invention, established human
cell lines which produce human antibodies, and human antibodies can
be obtained. The above-described human stem cells are transplanted
to the mouse of the present invention, and cells responsible for
immunity such as T-cells or B-cells are differentiated and
proliferated. Alternatively, cells responsible for immunity such as
human T-cells or B-cells are transplanted to the mouse and
engrafted in the mouse body, and thereby obtained is a mouse having
the cells responsible for human immunity and capable of producing
human antibodies. When human stem cells are transplanted, the
differentiation and proliferation of T-cells and B-cells are
realized in 6 to 8 weeks, enabling the production of human
antibodies.
[0071] By administering antigens to the mouse having human T-cells
and B-cells engrafted thereto and held, it is possible to obtain
human antibodies against the antigens and cells capable of
producing the antibodies. The administration of the antigens to the
mouse of the present invention may be carried out by the same
method as is conventionally used for immunizing a mouse.
[0072] Human antibody producing cells can be collected from each
organ of the mouse, especially the spleen, lymph nodes etc. When
the ratio of the human antibody producing cells is high, the
collected cells can directly be used for establishing a cell line.
However, when the ratio is low, if necessary, purification may be
carried out by e.g. the affinity column method using anti-human
B-cell antibodies. Further, it is desirable to eliminate mixed-in
mouse cells by e.g. cytolysis method using anti mouse antibodies
and complements.
[0073] The thus obtained human antibody producing cells are made
into a established cell line by a transformation method using
Epstein-Barr virus (EBV), a cell fusion method wherein the cells
are fused with suitable proliferation viable cells, or the like.
Then, obtainable is a human antibody-producing established cell
line capable of multiple passages while producing antibodies.
4. Production of a Pathologic Model Mouse with Tumor
[0074] In the mouse of the present invention, human tumors can be
engrafted and proliferated, and an animal model of a human tumor
can be obtained by transplanting tumor cells to the mouse of the
present invention. For example, the administration of the human
tumor cells causes the proliferation thereof inside the mouse body,
and thus a mouse having a human tumor can be obtained. Examples of
the cells to be used in this case include subcultured lines of
human tumor in a conventional nude mouse, and cell lines derived
from HTLV-1 leukemia such as ED-40515(-), MT-1 and TL-Oml. In
addition, human tumor tissues are chopped into pieces having a size
of several mm, and these cancer tissue pieces may directly be
transplanted and engrafted to the mouse of the present invention.
In this case, the site of the mouse to which tumor cells or tissues
are transplanted is not limited, but in the case of cells, they may
be transplanted intraperitoneally, intravenously or subcutaneously
to the mouse and in the case of the tissues, they may be
transplanted subcutaneously to the mouse. Any subcutaneous site of
the mouse may be acceptable such as subcutaneous gluteal region,
but it is desirable to transplant them subcutaneously at an auricle
or a dorsal region because the tumor can be checked without
incision. Further, in order to obtain results which reflect
clinical effects of an anti cancer agent, it is desirable to
transplant them at the identical site as that for clinical test (in
the case of colon cancer cells, the cells are to be transplanted to
the colon). When the cells are transplanted, a tumor is formed
within several weeks to several months. Specifically, when HTLV-1
cells are transplanted subcutaneously at a posterior auricle, a
tumor is formed in 2 weeks, thereby enabling expeditious production
of a practical tumor model mouse.
[0075] Moreover, when a tumor is transplanted to the mouse of the
present invention, metastases of tumor cells such as leukemic
changes are observed and the mouse is usable as a model animal for
tumor metastasis.
[0076] Using the thus obtained human tumor model mouse, screening
of an anti cancer agent, antimetastatic drug etc. can be performed.
As a method therefor, a candidate agent is administered to a mouse
having a tumor formed, by a suitable method e.g. oral, transdermal
administration or the like. Then, observation on the size of the
tumor, the size and number of metastatic focuses, the viability of
the mouse etc., allows judgment on the effect of the drug.
5. Production of a Viral Infectious Disease Model Mouse
[0077] Use of the mouse of the present invention enables the
obtainment of a viral infectious disease model mouse. Namely, by
transplanting to the mouse of the present invention cells which may
be infected with a human virus, and infecting the cells with the
virus, or transplanting cells infected with a virus, it is possible
to obtain a viral infectious disease model animal, which has
virus-infected cells engrafted and held.
[0078] In a conventional mouse, only an M-tropic virus which
infects macrophages can proliferate, but the proliferation of
T-tropic viruses such as HIV, HTLV-1, which infects T-cells becomes
possible using the mouse of the present invention.
[0079] For example, 1.times.10.sup.7 to 1.times.10.sup.8 of human
peripheral blood mononuclear leukocytes are intraperitoneally
administered to the mouse of the present invention, and after
several days some hundreds to thousands of TCID.sub.50 of HIV were
inoculated, thereby obtaining a HIV-infected model mouse having
human cells infected with HIV. The HIV infection can be detected
through the expression of HIV antigens such as p24 positive cells
as an index.
[0080] Instead of HIV, the inoculation of HTLV-1 enables the
obtainment of an HTLV-1 infected model mouse.
[0081] Use of the animal model for disease obtained according to
the present invention, allows in vivo research on proliferation
mechanisms of HIV, HTLV-1 etc., further development of therapies
for virus infections, screening of therapeutic products for virus
infection, or the like.
6. Production of a Mouse Having Enhanced Engraftment Capacity of
Heterologous Cells Using a NOG Mouse
[0082] Use of the NOG mouse of the present invention enables the
production of a mouse having more enhanced heterologous cell
engraftment. For example, such a mouse can be obtained by
backcrossing a mouse wherein a gene relating to the mouse immune
system is knocked out with the NOG mouse of the present invention.
Examples of the genes relating to the immune system include
cytokine receptor gene, cytokine gene etc.
[0083] Further, by introduction of human cytokine gene, which
relates to the differentiation and proliferation of human cells, or
the like (e.g. hGM-CSF or hSCF etc.), it is possible to produce a
mouse having more enhanced heterologous cell engraftment. For
instance, in accordance with a method of Pro. Natl. Acad. Sci. USA
77:7380-7384, 1980, or the like, the above gene is inserted into a
pronuclear fertilized egg of the mouse, and an individual having
this introduced gene incorporated thereinto is selected, thereby
producing a mouse which expresses a human cytokine gene etc. Then,
this mouse and the NOG mouse of the present invention were crossed
with each other, thereby producing a mouse having enhanced
heterologous cell engraftment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0084] FIG. 1 shows an outline of backcrossing for producing a NOG
mouse.
[0085] FIGS. 2A and 2B show time-course changes of human CD45
positive cells and human CD41 positive cells, after the
introduction thereof, in peripheral blood of the NOG mouse to which
CD34 positive cells are transplanted.
[0086] FIG. 3 shows the ratio of CD45 positive cells in the bone
marrow and spleen of the NOG mouse to which CD34 positive cells are
transplanted.
[0087] FIG. 4 shows the ratios of CD45 positive cells in peripheral
blood of mice in a comparative test between the NOG mouse and a
.beta.2 microglobulin deficient NOD-SCID mouse (NOD/LtSz-scid,
.beta. 2m null mouse).
[0088] FIGS. 5A and 5B show FACS patterns of NK cells and dendritic
cells in spleen cells obtained from each mouse strain.
[0089] FIGS. 6A, B and C show the results of ELISA for detecting
the production amount of cytokine under the stimulation of Listeria
monocytogenes antigens in spleen cells obtained from each mouse
strain.
[0090] FIG. 7 shows the removal of NK activity in the NOG mouse and
the NOD/LtSz-scid, .beta. 2m null mouse.
[0091] FIG. 8 shows the results of FACS wherein bone marrow cells
from primary, secondary and tertiary mice to which human CD34
positive cells have been transplanted, are stained with human
CD45.
[0092] FIGS. 9A and 9B show engraftment and differentiation of
human cells in the thymus of the NOG mouse to which human CD34
positive cells have been introduced.
[0093] FIGS. 10A and 10B show engraftment and differentiation of
human cells in the thymus of the NOG mouse to which human CD34
positive cells have been introduced.
[0094] FIGS. 11A and 11B show engraftment and differentiation of
human cells in the spleen of the NOG mouse to which human CD34
positive cells have been introduced.
[0095] FIGS. 12A and 12B show engraftment and differentiation of
human cells in the spleen of the NOG mouse to which human CD34
positive cells have been introduced.
[0096] FIGS. 13A and 13B show engraftment and differentiation of
human cells in peripheral blood of the NOG mouse to which human
CD34 positive cells have been introduced.
[0097] FIGS. 14A and 14B show engraftment and differentiation of
human cells in bone marrow of the NOG mouse to which human CD34
positive cells have been introduced.
[0098] FIGS. 15A, B and C show the ability of NOG mouse
transplanted with umbilical cord blood (CB), bone marrow (BM) and
peripheral blood stem cells (PBSC), respectively, to produce human
antibodies.
[0099] FIG. 16 shows the tumor formations after transplanting
LM-2-JCK to each mouse strain.
BEST MODE FOR CARRYING OUT THE INVENTION
[0100] Next, the present invention will be described in detail by
referring to Examples.
Example 1
Production of an Immunodeficient Mouse (NOG Mouse) with the
Deletion of NK Activity and Declined Dendritic Cell Function,
Examination of the Heterologous Cell Engraftment in the Mouse, and
Establishment of an Assay System of Human Stem Cells Using the
Mouse
(1) Production of an Immunodeficient Mouse (NOG Mouse) with the
Elimination of NK Activity and Reduced Dendritic Cell Function
[0101] In order to obtain multifunctional immunodeficient mice with
depleted NK activity, interleukin-2 receptor .gamma. chain knockout
mice (IL-2R.gamma.KO mice) (8 week-old) which were transferred from
Prof. Kazuo Sugamura (Department of Microbiology and Immunology,
Tohoku University, School of Medicine) were backcrossed with
NOD-Shi-scid mice (8 week-old) which were kept in the Central
Institute for Experimental Animals (also available from CLEA JAPAN,
INC.), and thereby F1 mice having an IL-2R.gamma. mutant gene
introduced thereinto were produced. The introduction of the mutant
IL-2R.gamma. chain gene in the F1 mice was confirmed by PCR
amplification and detection of the gene. Specifically, first, DNAs
were extracted by a DNA automatic extractor (MagExtractor
manufactured by TOYOBO) from 100 .mu.l of blood taken from ocular
fundus of the F1 mice. PCR buffer solution containing 23.5 L of 1.5
mM MgCl.sub.2, 0.4 mM dNTP and two sets of 25 pmol primers (the
following primers PI and PIII were used for the determination of
wild type, and a set of the following primers PI and PII were used
for the determination of mutant type) was added to 1.5 .mu.L of
this DNA, and PCR was conducted under the following amplification
conditions for the determination of whether IL-2R.gamma. chain
genes were wild type or mutant type.
TABLE-US-00001 (primers) PI: (SEQ ID NO: 1)
5'-CTGCTCAGAATGATGCCTCCAATTCC-3' PII: (SEQ ID NO: 2)
5'-CCTGCGTGCAATCCATCTTGTTCAAT-3' PIII: (SEQ ID NO: 3)
5'-GATCCAGATTGCCAAGGTGAGTAG-3'
[0102] (PCR Amplification Conditions)
[0103] The conditions were heating at 94.degree. C. for 5 minutes;
30 to 35 cycles of 1 minute at 94.degree. C., 1 minute at
55.degree. C., and 1 minute at 72.degree. C.; and thereafter
heating at 72.degree. C. for 10 minutes.
[0104] The PCR products obtained by the above PCR were subjected to
electrophoresis in 2% agarose gel, and measured according to the
size of the coloring band detected after ethidium bromide stain.
The sizes of the bands, about 660 bp for wild type and about 350 bp
for mutant type, were observed.
[0105] (Backcrossing)
[0106] Next, the F1 mice having the mutant IL-2R.gamma. gene
introduced thereinto were crossed with NOD/Shi-scid mice, thereby
obtaining F2 mice. Further, by detecting the introduction of the
mutant IL-2R .gamma. chain gene into the F2 mice in the same manner
as above, and detecting immunoglobulins in serum by an
immunodiffusion method, mouse individuals which had the mutant
IL-2R .gamma. chain gene and had a homozygous scid gene were
selected. Thereafter, the mouse individuals were crossed with
NOD/Shi-scid mice, and among the born mice, mice having mutant
IL-2R .gamma. chain gene were further crossed with NOD/Shi-scid
mice.
[0107] The above backcross was repeated at least 9 times, thereby
producing NOG (NOG) mice (FIG. 1 shows the outline). Here, since
the IL-2R .gamma. chain gene exists on an X chain chromosome, it is
effective to use male IL-2R.gamma.KO mice.
(2) Examination on Engraftment Capacity of Heterologous Cells in
NOG Mice
[0108] Next, using the NOG mice obtained by the above crossing and
conventional immunodeficient mice, NOD/Shi-scid mice, examinations
were made on the level of impact that anti-NK antibody treatment
has on engraftment of heterologous cells in these mice.
[0109] (Anti-IL-2 Receptor .beta. Chain Monoclonal Antibody)
[0110] Anti-IL-2 receptor .beta. chain monoclonal antibodies (clone
TM .beta.1) were produced from hybridomas produced and provided by
Prof. Masayuki Miyasaka, School of Medicine, Osaka University
(Tanaka T, Tsudo M, Karasuyama H et al., A novel monoclonal
antibody against murine IL-2Receptor beta-chain. Characterization
on of receptor expression in normal lymphoid cells and EL-4 cells.
J Immunol 1991; 147(7):2222-8). In particular, the hybridomas were
intraperitoneally administered to BALB/cA-nu mice and collected
from ascites after several weeks.
[0111] One mg per mouse of the antibodies was intraperitoneally
administered to five NOD/Shi-scid mice (8 to 12 week-old) and three
NOG mice (8 to 12 week-old). Further, as controls with no
administration of the antibodies, physiological saline was
administered to four NOG mice (8 to 12 week-old).
[0112] Moreover, human peripheral blood lymphocytes were collected
by use of density gradient centrifugation using Lymphoprep, from
blood taken from volunteers.
[0113] 1.times.10.sup.7 of the obtained human peripheral blood
mononuclear leukocytes were intraperitoneally administered to the
above mice on the second day after the administration of TM
.beta.1.
[0114] The mice were sacrificed 2 weeks after the administration of
human peripheral blood lymphocytes, and the ascites containing the
whole peritoneal exudates cells were fully washed with RPMI-1640
culture medium and thereby collected. Out of all the peritoneal
cells, all the peritoneal exudates cells were counted by flow
cytometry, and depending on their amounts, the engraftment and
proliferation of human cells into mice were determined. The same
operations were conducted for control mice with no administration
of the antibodies. Their results are shown in Table 1.
TABLE-US-00002 TABLE 1 Impact of anti-NK antibodies (TM.beta.1) on
engraftment and proliferation of human peripheral blood mononuclear
leukocytes in NOD/Shi-scid mice and NOG mice Number of collected
cells Human cells % TM.beta.1 Number of (.times.10.sup.6, HLA+
Mouse strain treatment mice distribution) (distribution) hCD4.sup.+
hCD8.sup.+ NOD/Shi-scid + 5 4.8 41.1 ND ND (2.77-6.8) (4.2-65) NOG
+ 3 11.1 61.9 19.8 28.2 (8.3-15.0) (47.4-74.6) - 4 11.2 63.3 34.3
25.9 (7.9-20) (51.4-69.7)
[0115] It is clear from the results shown in Table 1 that extremely
high ratios of human cells were differentiated, engrafted and
proliferated, even without the treatment of the antibodies, in NOG
mice to which human peripheral blood mononuclear leukocytes were
introduced, as compared with conventional TM .beta.1 treated
mice.
[0116] (Anti Asialo-GM1 Antibody)
[0117] Next, using NOG mice and NOD/Shi-scid mice, examinations
were made on the level of impact, which would be given by anti
asialo-GM1 antibodies (AGM1) (rabbit) (Wako Pure Chemical
Industries, Ltd., 014-09801), on engraftment capacity of cells
derived from human umbilical cord blood in these mice.
[0118] First, 2.4 Gy of X ray were irradiated on NOG mice (8 to 12
week-old) and NOD/Shi-scid mice (8 to 12 week-old) which were
numbered as indicated in Table 2. Then, 20 .mu.L per mouse of anti
asialo-GM1 antibodies diluted with 400 .mu.L PBS were administered
to these mice just before the administration of the cells derived
from human umbilical cord blood. Also, physiological saline was
intraperitoneally administered to control mice (No. 5, 6, 10, 11,
15, 16 mice in Table 2) without administration of the
antibodies.
[0119] With respect to human umbilical cord blood CD34 positive
cells, mononuclear leukocytes were collected by Ficoll-Hypaque
gradient centrifugation from umbilical cord blood taken from
volunteers from whom approval was each obtained in advance.
Further, CD34 positive cells were isolated by Dynabeads M-450 CD34
and DETACHaBEAD CD34 (Dynal As, Oslo, Norway). (Ueda T, Tsuji K,
Yoshino H et al., Expansion of human NOD/SCID-repopulating cells by
stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and
soluble IL-6 receptor. J Clin Invest 2000; 105(7): 1013-21)
[0120] 1.times.10.sup.5 of the obtained CD34 positive cells derived
from human umbilical cord blood were introduced through tail veins
of the mice immediately after the administration of anti asialo-GM1
antibodies.
[0121] The mice were sacrificed 4 weeks after the administration of
CD34 positive cells derived from human umbilical cord blood, their
peripheral blood was collected, and human cells (CD45.sup.+) in
mononuclear leukocytes and human platelets (CD41.sup.+) in the
whole blood were counted by flow cytometry. Depending on their
amounts, the engraftment capacity and proliferation of human cells
into mice were determined. The same operations were conducted for
control mice without administration of the antibodies. Their
results are shown in Table 2.
TABLE-US-00003 TABLE 2 Differentiation and proliferation of
introduced human umbilical cord blood CD34.sup.+ in NOG mice No. of
4 weeks after introduction introduced No. of No. of No.of AGM1
CD34.sup.+ Days after Leukocytes erythrocytes platelets hCD45.sup.+
hCD45.sup.+ hCD41.sup.+ hCD41.sup.+ Mouse No. Mouse Strain
treatment cells transplant (10.sup.2/ul) (10.sup.4/ul)
(10.sup.4/ul) (%) (/ul) (%) (/ul) 1 NOD/Shi-scid + 100000 31(4 wk)
12 782 134 0.19 2 0.001 20 2 NOD/Shi-scid + 100000 31(4 wk) 12 809
147 0.94 11 0.022 318 3 NOD/Shi-scid - 100000 31(4 wk) 10 758 104
0.48 5 0.004 41 4 NOD/Shi-scid - 100000 31(4 wk) 14 773 109 3.24 45
0.166 1814 5 NOG - 100000 31(4 wk) 12 713 89.3 5.84 70 0.255 2274 6
NOG - 100000 31(4 wk) 17 749 108 9.29 158 0.324 3498 7 NOD/Shi-scid
+ 100000 29(4 wk) 10 734 139 1.82 18 0.056 772 8 NOD/Shi-scid +
100000 29(4 wk) 10 787 118 1.51 15 0.094 1105 9 NOD/Shi-scid -
100000 29(4 wk) 7 722 132 0.97 7 0.059 776 10 NOG - 100000 29(4 wk)
36 602 218.1 1.84 66 0.170 3709 11 NOG - 100000 29(4 wk) 10 725 111
9.28 93 0.518 5748 12 NOD/Shi-scid + 100000 28(4 wk) 7 696 92.4
1.74 12 0.007 63 13 NOD/Shi-scid + 100000 28(4 wk) 10 787 96 0.40 4
0.008 73 14 NOD/Shi-scid - 100000 28(4 wk) 10 710 82.6 0.41 4 0.021
173 15 NOG - 100000 28(4 wk) 2 684 103 8.52 17 0.101 1040 16 NOG -
100000 28(4 wk) 2 773 86.5 14.98 30 0.196 1692 Average
NOD/Shi-scid(AGM1+) 1.10 10.52 0.03 391.74 NOD/Shi-scid(AGM1-) 1.27
15.25 0.06 701.14 NOG 7.11 61.99 0.22 2565.96
[0122] It is clear from the results shown in Table 2 that extremely
high ratios of human cells were differentiated, engrafted and
proliferated, even without the treatment of the antibodies, in NOG
mice to which CD34 positive cells derived from human umbilical cord
blood were introduced, as compared with conventional mice which
were treated with anti asialo-GM1 antibody.
(3) Establishment of Assay System of Human Stem Cells Using NOG
Mice
[0123] Mice to be used here were NOD/Shi-scid mice and NOG mice of
the present invention. 1.times.10.sup.5 of CD34 positive cells
taken from human umbilical cord blood (CB) were transplanted to the
above mice which had received 2.4 Gy of radiation.
[0124] Human cells in peripheral blood, bone marrow, spleens, and
thymuses were analyzed by FACS. The ratios of CD45 positive cells
in peripheral blood 8 weeks after the transplant were 37% for NOG
mice and 7% for NOD/Shi-scid mice. In bone marrow, the ratios were
65% and 20%, respectively. In addition to a high ratio of
chimerism, it was found that differentiations occurred into various
cell lines including B and T lymphocytes in peripheral blood, bone
marrow, spleens, and thymuses of the NOG mice 3 month after the
transplant. It was observed that human CD33.sup.+ bone marrow
cells, CD 19.sup.+B cells, CD3.sup.+T cells, CD56.sup.+NK cells,
CD41a.sup.+huge nucleoplasm, and glycophorin A.sup.+ erythrocytes
were present in bone marrow. Further, it is interesting to note
that though there were observed a small number of human T cells in
the thymuses, CD4.sup.+/CD8.sup.- T cells, CD4.sup.-/CD8.sup.+T
cells, and CD4.sup.+/CD8.sup.+ T cells were observed in the
spleens. This indicates that human hematopoietic stem cells are
differentiated and proliferated into mature T cells at sites apart
from the thymus.
[0125] Umbilical cord blood was obtained at birth of a healthy
newborn from a normal pregnant mother from whom approval was
obtained in advance. The umbilical cord blood was heparinized and
preserved, and treated by the operation described below within 24
hours after collecting. Then, it was used for a transplant
test.
[0126] The purification of CD34 positive cells was conducted as
follows. The heparinized umbilical cord blood was diluted two times
with a buffer solution prepared by mixing phosphate buffered saline
with 5% fetal bovine serum, and thereafter mononuclear leukocytes
were isolated using Ficoll. From the isolated mononuclear
leukocytes, CD34 positive cells were purified by use of
Dynabeads.TM. M-450 CD34 available from Dynal Biotech, Ltd.
Although the method therefor is as instructed by Dynal Biotech,
Ltd., it is summarized as follows. Using phosphate buffered
physiological saline containing 2% bovine serum albumin and sodium
citrate, the concentration of the mononuclear leukocytes was
adjusted so as to be 4.times.10.sup.7/mL. 100 .mu.L of
well-suspended Dynabeads CD34 was added per 1 mL of mononuclear
leukocyte suspension, and they were blended and reacted with each
other over ice for 30 minutes. Beads which formed rosettes with
cells were collected using a magnet. Subsequently, to the Beads
forming rosettes with cells, a required amount of Detachabead.TM.
CD34 was added and reacted therewith, blending at 37.degree. C. for
15 minutes. As CD34 positive cells were liberated from the Beads, a
magnet was used to remove only the Beads, thereby obtaining CD34
positive cells.
[0127] For transplanting, 8 to 12 week-old NOD/Shi-scid mice, NOG
mice, and NOD/LtSz-scid, .beta.2m null mice, all raised in an SPF
(specific pathogen free) environment were used. These mice were
raised in the animal experiment facility of Kyoto University,
School of Medicine under the regulations of the facility.
[0128] In a comparative test for the former 2 types of mice, the
mice were twice irradiated with 1.2 Gy gamma ray (gamma cell
.sup.137Cs), and 100,000 of CD34 positive cells per mouse were
transplanted through the tail vein. For the NOD/Shi-scid mice, 200
.mu.g of anti asialo GM1 antibodies (Wako Pure Chemical Industries,
Ltd.) were intraperitoneally administered immediately before the
transplant and every eleventh day after the transplant.
[0129] In a comparative test between NOG mice and NOD/LtSz-scid,
.beta.2m null mice, the mice were twice irradiated with 1.2 Gy
gamma ray in the same way as above, and thereafter 40,000 or 10,000
of CD34 positive cells were transplanted through the tail vein.
[0130] After transplantation, 732 mg/L of neomycin sulfate were
added to the mice's drinking water for protection against
infection.
[0131] The mice were ether-anaesthetized at moments as indicated,
and then peripheral blood was collected from the orbital venous
plexus for measuring the positive ratio of human hemocytes by use
of flow cytometry. Although the flow cytometry was conducted in
accordance with a widely-used general method, it is summarized as
follows. The collected blood was instantly blended well with
EDTA-2Na and allowed to stand at room temperature until analysis.
An optimum amount of antibodies was added to and reacted with 50 to
100 .mu.L of the whole blood at 4.degree. C. for 30 minutes.
Thereafter, hemolysis and immobilization were effected using FACS
solution (Becton Dickinson and Co.), and the ratio was measured
using FACS Calibur (Becton Dickinson and Co.). In the case of bone
marrow and spleen, the mice were sacrificed with cervical vertebra
dislocation and femora and spleens were eviscerated. Then, bone
marrow and splenic cells were each liberated in culture solution
containing 5% fetal bovine serum, and thereafter as cell
suspensions, they were treated in the same manner as peripheral
blood and analyzed by flow cytometry.
[0132] Antibodies to be used for the analysis were FITC conjugated
anti human CD45 antibodies, PE conjugated anti human CD10
antibodies, PE conjugated anti human CD33 antibodies, PE conjugated
anti human CD3 antibodies, PE conjugated anti human CD34
antibodies, PE conjugated anti human CD41 antibodies (Beckton,
Dickinson and Co.), PC5 conjugated anti human CD38 antibodies, PC5
conjugated anti human CD56 antibodies, PC5 conjugated anti human
CD19 antibodies (Immunotech), APC conjugated anti mouse CD45
antibodies, and FITC conjugated anti mouse CD41 antibodies (BD
Pharmingen).
[0133] With respect to human CD45 positive cells and human CD41
positive cells in mouse peripheral blood, changes in the ratios (%)
and the absolute numbers (/.mu.L) were investigated every fourth
week until the 12th week (FIG. 2A and FIG. 2B). As shown in FIGS.
2A and 2B, significantly high ratios and absolute numbers were
observed for NOG mice. The mice were sacrificed from 12 weeks
onward after the transplant, and when the ratios of human CD45
positive cells in bone marrow and spleens were investigated in the
same way as above, the NOG mice had very high positive ratios.
(FIG. 3)
[0134] In the comparative test between NOG mice and NOD/LtSz-scid,
.beta.2m null mice, the positive ratios of human CD45 positive
cells in mouse peripheral blood were investigated (FIG. 4). As
shown in FIG. 4, the NOG mice had a higher positive ratio of human
cells and exhibited a better engraftment capacity of human cells
than NOD/LtSz-scid, .beta.2m null mice.
Example 2
Examinations on Functional Incompetence of Dendritic Cells of NOG
Mice in NK Activity and Cytokine Production
[0135] Three female NOG mice (10 to 12 week-old) of the present
invention, four female and four male NOD/Shi-scid mice (10 to 12
week-old) obtained by backcrossing C.B-17-scid mice with NOD/Shi
mice, and two female and two male NOD/LtSz-scid, .beta.2m null mice
(10 to 12 week-old) (or referred to as null .beta.
2m(null)NOD/LtSz-SCID, .beta.2m microglobulin deficient NOD/SCID
mice; produced by Dr. Shultz, L. D. et al. of The Jackson
Laboratory Kollet O, Peled A, Byk T et al., beta2
microglobulin-deficient (.beta.2m(null))NOD/SHI-SCID mice are
excellent recipients for studying human stem cell function. Blood
2000; 95(10):3102-5) were used for the following examinations.
[0136] After the NOD/Shi-scid mice were treated with anti asialo
GM1 antibodies (.alpha.AGM), spleen cells were collected. The
obtained cells were divided into two groups, and CD11c antigen
positive cells (regarded as dendritic cells) were removed from one
of them using a magnetic cell sorter (MACS). Likewise, splenic
cells were collected from non-treated NOD/LtSz-scid, .beta.2m null
mice and NOG mice. A small amount of these cells was taken from
each of them and provided for FACS analysis. Further, these four
kinds of cells were divided into three groups: group I: NOG mice (3
mice); group II: AGM non-treated NOD/Shi-scid mice (2 males),
.alpha.AGM treated NOD/Shi-scid mice (3 mice), and NOD/Shi-scid
mice (3 mice) which were treated with .alpha.AGM and further from
which CD11c were removed; and group III: NOD/LtSz-scid, .beta.2m
null mice (4 mice). They were adjusted to have a cell concentration
of 1.times.10.sup.7/ml in RPMI-1640, and 100 .mu.l of each of them
were dispensed into a well, stimulated by an equivalent amount of
Listeria monocytogenes (LM) antigens, and cultured (using
Triplicate). Since LM is 10.sup.11 Units/mL, it was diluted for use
so as to be 2.times.10.sup.7 Units/mL with RPMI-1640. In this case,
specimens with no LM addition were used as a control. After 24
hours, the supernatants were collected, and the amounts of cytokine
such as IFN-.gamma. were determined by ELISA.
[0137] Subclasses (in particular, CD11c, CD11b) in spleen cells
were analyzed by FACS.
[0138] FITC-labeled CD3 and biotine-labeled Pan NK(DX5) were used
as a T-cell marker and an NK cell marker, respectively.
FITC-labeled anti-CD 11c and PE-labeled anti-CD 11b were used as a
macrophage and dendritic cell marker.
[0139] Further, NK activity in NOG, C.B-17-scid, NOD/Shi-scid, and
NOD/LtSz-scid, .beta.2m null mice (10 to 12 week-old) was examined
by cytotoxicity test targeting .sup.51 Cr-labeled NK susceptible
YAC-1 cells. Namely, splenic cells were isolated from the mice and
mixed and cultured with .sup.51Cr-labeled YAC-1 cells at various
ratios therebetween. After culturing at 37.degree. C. for 4 hours
under 5% CO.sub.2, the radioactivity in supernatants was measured
by a liquid scintillation counter. The NK activity is indicated
according to the following calculation method.
% specific cytotoxicity=(specific radioactivity-background
radioactivity)/(maximum radioactivity-background
radioactivity).times.100
[0140] FIGS. 5A and 5B show FACS patterns of the spleen cells
obtained from each mouse strain.
[0141] NK cells were detected from NOD/Shi-scid and NOD/LtSz-scid,
.beta.2m null mice, though NK cells were not detected at all from
NOD/Shi-scid mice treated with anti asialo GM1 antibody and NOG
mice. CD11c positive cells, which were used as a dendritic cell
marker were detected from all the mice at extremely high ratios. It
was confirmed that CD11c positive cells were almost completely
removed from the splenic cells of the NOD/Shi-scid mice treated
with anti asialo GM1 antibody by magnetic beads.
[0142] FIGS. 6A, 6B and 6C show detection results by ELISA on the
production amount of cytokine in the above splenic cells under the
LM stimulation. The following was found: although IFN .gamma.
production was observed in NOD/Shi-scid mice not treated with anti
asialo GM1 antibody and NOD/LtSz-scid, .beta.2m null mice, there
was no detection from NOG mice; and, as with NOG mice, by removing
CD11c positive cells from the NOD/Shi-scid mice treated with anti
asialo GM1 antibody, IFN .gamma. production was not observed.
[0143] FIG. 7 shows NK activity of the splenic cells obtained from
each mouse strain.
[0144] In comparison with C.B-17-scid mice, reduction of NK
activity in NOD/Shi-scid mice was observed, but absolutely no NK
activity was observed in NOG mice and NOD/LtSz-scid, .beta.2m null
mice.
[0145] In view of the foregoing, it became clear that NOG mice and
NOD/LtSz-scid, .beta.2m null mice completely lost their NK
activity. However, according to the result of FACS analysis, it
became clear that the NOG mice lost NK cells, though the
NOD/LtSz-scid, .beta.2m null mice had NK cells present therein but
lost NK activity.
[0146] Further, it was revealed that the functional incompetence of
the dendritic cells was a cause for the cytokine production decline
in splenic cells, which was observed in the NOG mice. On the other
hand, it was indicated that the NOD/LtSz-scid, .beta.2m null mice
had almost the same patterns of FACS and cytokine production as the
anti asialo GM1 antibody-treated NOD/Shi-scid mice. It was obvious
that the NOD/LtSz-scid, .beta.2m null mice lost NK cells and their
dendritic cells were normal.
Example 3
Successive Transplant of Human Stem Cells Using NOG Mice
[0147] In order to examine self-replicability of gene-introduced
human stem cells, umbilical cord blood stem cells were transplanted
to NOG mice, and it was examined whether or not secondary or
tertiary transplant is possible.
[0148] Umbilical cord blood was obtained from pregnant women, with
their consent and approval, for use in tests. After mononuclear
cells were isolated from umbilical cord blood using Ficoll-Hypaque
(Lymphoprep, 1.077.+-.0.001 g/ml; Nycomed, Oslo, Norway), CD34
positive cells were purified using a CD34 positive separation
column (MACS, Miltenyi Biotec, Glodbach, Germany). The CD34
positive cells had a purity of 96.+-.3%.
[0149] Lentivirus vector pCS-CG was a vector which enables a GFP
gene to be expressed by a CMP promoter, and it was provided by Dr.
Miyoshi (Immunology, Medical Branch, University of Tsukuba). After
umbilical cord blood CD34 positive cells were cultured for 24 hours
in serum-free medium StemPro TM-34SFM (Gibco BRL) containing 50
ng/mL of each TPO, SCF and Flk-2/Flt-3 ligand (FL), they were
subjected to five-hour infection of recombinant lentivirus CS-CG by
MOI30 (Kawada H et al., Exp. Hematol. 27:904-915, 1999).
Thereafter, cells were washed and subjected to 5-day extracorporeal
amplification culture in the same serum-free medium as above in the
presence of murine bone marrow stroma cells HESS-5 (Oki M et al.,
Exp. Hematol. In press. 2001).
[0150] After 7 week-old NOD/Shi-scid mice were subjected to 3 Gy
radiation, 3.times.10.sup.5 of gene-introduced cells which were
amplified by culture, were introduced through the tail vein. After
6 weeks, surface antigens of murine bone marrow cells were
analyzed, and 1.times.10.sup.7 of cells were introduced into
irradiated NOG mice (secondary transplant). After 6 weeks, the same
analysis was conducted, and 1.times.10.sup.7 of cells were
introduced into irradiated NOG mice (tertiary transplant). Further,
after 6 weeks, the same analysis was conducted.
[0151] The analysis of surface antigens was conducted using FACS
Calibur (Becton, Dickinson and Co.). The antibodies used here were
FITC-labeled anti-human CD45 (T-200), PE-labeled anti human CD2
(39C1.5), CD3(UCHT1), CD4(SK3), CD14(LeuM3), CD19(4G7), CD20(2H7),
CD33(WM53), CD41(P2), CD56(N901), and glycophorin A (KC16), and all
of them were purchased from Becton, Dickinson and Co.
[0152] The gene introduction efficiency into CD34 positive cells
was 40.+-.5% (n=5). FIG. 8 shows the results of FACS wherein mouse
bone marrow cells of primary, secondary, and tertiary transplant
mice were stained with human CD45. It was observed that
gene-introduced human cells existed in all the mice.
[0153] By using NOG mice of the present invention as a host, the
inventors became the first in the world to successfully accomplish
the introduction of a gene capable of being expressed in
hematopoietic stem cells which can be transplanted to up to a
tertiary host. Use of conventional NOD-scid mice enabled up to
secondary transplant (Guenechea G. et al., Nature Immunol, 2,
75-82, 2001), and beyond this it was shown that the present mice
were more sensitive and useful as an assay system for hematopoietic
stem cells.
Example 4
Production of Complete Human Antibodies Using NOG Mice
[0154] After NOG mice (8 week-old) and NOD/Shi-scid mice (8
week-old) were irradiated with 2.5 Gy and 3.5 Gy, respectively,
they were subjected to intravenous transplant of 1.times.10.sup.6
of CD34.sup.+ cells which were purified with magnet beads from
human umbilical cord blood (provided by Tokai University, Cell
Transplant Research Center, with the consent and approval of the
pregnant women). Specifically, MNC was isolated from umbilical cord
blood using Ficoll, and then CD34.sup.+ cells were isolated by MACS
immunomagnetic separation system (Miltenyl Biotec, Glodach,
Germany). After confirming that the CD34.sup.+ cells had 97% or
more purity by FACS, they were used for transplant.
[0155] Blood, having been chronologically taken from orbits, and
after MNC fraction was obtained by Ficoll, was stained with
fluorescence-labeled CD45, CD19, and CD3 antibodies (Becton
Dickinson, San Jose, Calif.). The reconstruction of human
lymphocytes, and the ratios of B-cells and T-cells were confirmed
with CD45, and fluorescence-labeled CD19 and CD3, respectively, by
FACS.
[0156] Moreover, thymuses and spleens were extracted from these
mice, cells were prepared therefrom, the cell numbers were counted,
and they were stained with various fluorescence antibodies (Becton
Dickinson, San Jose, Calif.). Then, they were analyzed by FACS in
the same manner as the above reconstruction confirmation.
[0157] From 6 or 8 weeks after the transplant, antigens had been
administered to these mice. As an antigen, DNP-KLH was used. The
immunization was performed intraperitoneally with 100 .mu.g/mouse
of DNP-KLH together with an adjuvant of aluminum hydroxide (ALUM).
The same immunization was performed every two weeks and serum was
taken for measuring antibody titer by ELISA.
[0158] At this juncture, ELISA was performed as follows.
[0159] 96-well plate was coated with anti human IGs (ICN, Aurora,
Ohio) or DNP-KLH, and blocking and washing were performed with 3%
BSA. Diluted anti serum was added thereto. After the reaction at
room temperature for 2 hours, washing was performed. Then,
biotinylated anti-human IgM or anti-human IgG monoclonal antibodies
(Phermingen, San Diego, Calif.) were added. After the reaction at
37.degree. C. for 2 hours, washing was performed. Then,
avidinylated peroxidase was added for 1 hour reaction at room
temperature. The plate was washed and TMB peroxidase EIA substrate
kit solution (Bio-Rad Laboratories, USA) was added for 30-minute
reaction at room temperature. Then, the reaction was inhibited with
10% HCl, and the absorbance was measured at 450 nm. The Ig
concentration was calculated in accordance with the standard
curve.
(1) Efficiency of Transplanting and Reconstituting CD34.sup.+ Cells
Derived from Umbilical Cord Blood
[0160] With respect to the ratio of human CD45.sup.+ cells in the
NOG mice after transplanting the umbilical cord blood cells, the
ratio of human CD45 in peripheral blood was gradually decreased
from 4 weeks onward. However, from 12 weeks onward, suddenly the
ratio of increase of T-cells became high, and along with this, it
was observed that the ratio of human CD45 was also increased in
some mice (Table 3). Increases of the CD45.sup.+ ratio, caused by
such T-cell increase, had never been observed in peripheral blood
of the existing NOD/Shi-scid mouse. These NOG mice had never been
affected with GVHD until 14th week.
TABLE-US-00004 TABLE 3 Reconstitution of human cells in NOG mice:
peripheral blood SCT CD34.sup.+ 4 weeks 6 weeks 8 weeks Gp. Source
cells % CD45 % CD19 % CD3 % CD45 % CD19 % CD3 % CD45 % CD19 % CD3
2.sup.nd SCT CB2 freeze 1.0 .times. 10.sup.6 47.3 45.7 1.3 N.A.
N.A. N.A. 27.5 42.7 7.2 thawing PBSC1 freeze 1.8 .times. 10.sup.6
79.5 0.7 13.4 72.5 22.9 47.7 13.3 14.3 65.7 thawing PBSC2 freeze
1.8 .times. 10.sup.6 30.8 3.0 0.1 62.0 63.2 3.8 68.5 93.6 2.1
thawing PBSC3 freeze 1.8 .times. 10.sup.6 26.9 3.7 0.0 N.A. N.A.
N.A. 43.5 62.0 7.6 thawing 3.sup.rd SCT CB fresh 1.8 .times.
10.sup.5 4.8 5.7 0.1 82.2 95.9 0.0 12.4 79.1 1.0 BM freeze 1.9
.times. 10.sup.5 1.7 0.0 0.0 8.2 48.2 0.0 13.5 49.8 0.0 thawing
PBSC2 freeze 1.65 .times. 10.sup.6 1.4 2.0 0.0 51.6 91.7 0.0 25.6
91.4 0.0 thawing PBSC3 freeze 1.65 .times. 10.sup.6 2.1 1.3 0.2
24.1 88.4 0.0 45.3 90.2 0.0 thawing 4th SCT PBSC1 freeze 5.0
.times. 10.sup.5 19.4 2.6 0.0 27.7 39.8 0.0 39.2 92.4 0.1 thawing
PBSC2 freeze 5.0 .times. 10.sup.5 14.5 1.8 0.1 20.0 17.4 0.2 33.4
82.1 0.0 thawing PBSC3 freeze 5.0 .times. 10.sup.5 22.1 3.3 0.0
17.5 49.1 0.1 26.1 83.4 0.1 thawing 5.sup.th SCT BM freeze 1.5
.times. 10.sup.5 0.9 * * 0.2 * * thawing 6.sup.th SCT BMI fresh 3.6
.times. 10.sup.5 BM2 fresh 3.6 .times. 10.sup.5 PBSC1 freeze 9.0
.times. 10.sup.5 thawing PBSC2 freeze 9.0 .times. 10.sup.5 thawings
PBSC3 freeze 9.0 .times. 10.sup.5 thawing SCT 12 weeks 10 weeks Gp.
Source % CD45 % CD19 % CD3 % CD45 % CD19 % CD3 remarks 2.sup.nd SCT
CB2 19.8 27.9 4.9 PBSC1 Analyzed because of death at 8-week PBSC2
13.7 64.5 23.9 10.3 48.4 32.6 PBSC3 28.4 34.1 11.1 22.0 35.5 14.0
3.sup.rd SCT CB 21.8 71.7 10.1 BM 7.0 73.7 0.0 mixed PBSC2 25.8
85.6 0.1 PBSC3 14.3 82.5 0.0 4th SCT PBSC1 PBSC2 PBSC3 5.sup.th SCT
BM mixed 6.sup.th SCT BMI BM2 PBSC1 CD3 eliminated PBSC2 CD3
eliminated PBSC3 CD3 eliminated
(2) T-Cell Differentiation in NOG Mice
A. T-Cell Differentiation in the Thymus
[0161] FIGS. 9A, 9B, 10A and 10B show T-cell differentiation
patterns in thymuses. As shown in the figures, CD3 positive cells
were differentiated in the thymuses of these mice, and it was
indicated that these cells contained CD4/CD8 DN (Double negative),
DP (Double positive), and SP (Single positive) as well as those
cells in a normal thymus or detected by hu/m-RTOC, and a part of
them were mature to be CD1a-low T-cells in 11th or 13th week after
transplanting. The number of the thymic cells was 1 to
2.times.10.sup.6 and this was about one-hundredth of the number of
thymic cells in a normal mouse. This phenomenon was observed also
in NOD/Shi-scid mice, but its frequency was remarkably low, such as
8/28 (28.6%). As opposed to this, NOG mice had a frequency of 7/7
(100%).
B. T-Cells at Peripheries
[0162] FIGS. 11A, 11B, 12A, 12B, 13A, and 13B show further analysis
on T-cells in splenic cells or peripheral blood. CD3 positive cells
were observed even at the peripheries of the mice from which T-cell
differentiation was observed in their thymuses. These are a group
of CD4/CD8 positive cells, and this indicated that there was the
possibility that they included cells differentiated in
thymuses.
(3) B-Cell Differentiation in NOG Mice
A. Subset Differentiation of B-Cells
[0163] Also, FIGS. 14A and 14B show the expression ratios of CD5 by
B-cells among bone marrow cells. In bone marrow, the
differentiation of CD5 positive cell, so-called B1a cells, was not
facilitated, and groups of CD19 positive or IgM positive cells were
dominant. Further, cells in a group in which IgM is highly positive
expressed CD20. No abnormality was detected on the differentiation
of human B-cells in the bone marrow of these mice. This phenomenon
is also observed in B-cell differentiation in NOD/Shi-scid mice,
and thus it is considered a common quality.
B. B-Cell at Peripheries
[0164] FIGS. 13A and 13B show FACS patterns of B-cells at their
peripheries. The ratios of B-cells were almost the same as those in
NOD/Shi-scid mice. However, in spleens, CD5 positive cells,
so-called B1a cells which was a subgroup of B-cells were dominant,
and a large difference was detected from differentiation patterns
in bone marrow. On the other hand, IgM positive CD5 negative cell
groups were also detected at a ratio of about 20% and thus this
proved that cells, other than B1a cells, were also detected.
(4) Antibody Production Ability in NOG Mice
[0165] DNP-KLH was administered as an antigen to these mice, and
IgM and IgG antibody production amounts were chronologically
measured. As a result, antigen non-specific IgM and IgG antigen
specific IgM were detected by repeating the administration three
times. Although T-cells were detected in thymuses and peripheries,
antigen specific IgG production was not detected. (FIGS. 15A, 15B
and 15C) FIGS. 15A, 15B and 15C also show the results of mice
having transplanted thereto CD34.sup.+ cells derived from bone
marrow (BM) (FIG. 15B) and peripheral blood (PBSC) (FIG. 15C). In
bone marrow and peripheral blood, there was observed a tendency of
lower productions of specific IgM and non-specific IgG as compared
with the production in umbilical cord blood.
[0166] The above results indicate that the present mouse
differentiates human T-cells and B-cells and is valuable for use in
human antibody production systems.
Example 5
Neoplasm Proliferation System
[0167] NOD/Shi-scid mice, NOG mice, BALB/cAJc1-nu mice (purchased
from CLEA), and C.B-17/Icr-scid mice (purchased from CLEA) were
used. All the used mice were 5 weeks old or older.
[0168] As cells to be transplanted to the mice, transplant human
tumor cell line, LM-2-JCK was used. LM-2-JCK was a cell line which
was established from lymphoblast lymphoma of a 13-year-old female
patient and maintained by successive heterografts to nude mice. It
has been reported that though LM-2JCK expresses T-cell antigen CD4
and CD5, it does not express other cell antigens including
antibodies.
[0169] A solid tumor which was subcutaneously passaged 12 times in
nude mice was used for heterograft assay. The tumor was chopped
into pieces in F-10 nutrition supplementary medium (GIBCO BRL) by
scissors and fully dispersed by a pipette, and then cell suspension
was prepared by passing through a nylon mesh. The concentration of
viable cells in the suspension was calculated by trypan blue stain
(GIBCO BRL). After centrifugation, tumor cells were dispersed at
concentrations of 1.times.10.sup.7 and 1.times.10.sup.6 viable
cells/ml into physiological saline. The tumor cell dispersion
liquid was subcutaneously injected, in an amount of 0.1 mL, at both
flanks of mice by using 1 ml syringe equipped with 25 gage needle.
After the injection of the tumor cells, the size of the tumor and
the body weight were measured at intervals of one week. On the 21st
day after the 1.times.10.sup.6 cells were transplanted, when the
tumor became large, the mice were sacrificed and the weights and
sizes of the tumors were measured.
[0170] The difference in transplantability of LM-2-JCK among the
mice having different backgrounds is shown in Table 4.
TABLE-US-00005 TABLE 4 Heterotransplantation of LM-2JCK to mice
having different genetic backgrounds No. of tumor-engrafted No. of
cells Observation mice/No. of Mouse strain grafted period
transplanted mice Weight of tumor a) NOG 10.sup.6 21 days 10/10
(100%) 3.97 .+-. 2.10 b) NOD/Shi-scid 10.sup.6 21 days 10/10/(100%)
1.34 .+-. 0.77 C.B-17/Icr-scid 10.sup.6 21 days 8/10 (80%) 1.21
.+-. 1.06 NOG 10.sup.5 <9 weeks 8/8 (100%)c) n.t. NOD/Shi-scid
10.sup.5 <9 weeks 5/10 (50%) n.t. C.B-17/Icr-scid 10.sup.5 <9
weeks 0/10 (0%) n.t. a) The weight of tumor was measured 21 days
after the transplanting and indicated with average .+-. SD (g). b)
As to NOD/Shi-scid and C.B-17/Icr-scid, when p < 0.05 (t-test),
the significant difference is observed. c)As to NOD/Shi-scid (p
< 0.05) and C.B-17/Icr-scid (p < 0.01), the significant
difference is observed. (.chi..sup.2 test) n.t.: not tested
[0171] In the case of the transplant of 1.times.10.sup.6 cells, all
the tumors were engrafted in NOG mice and NOD/Shi-scid mice. In
contrast, the engraftment ratio for C.B-17/Icr-scid was 80%.
[0172] The tumor engraftment ratios in these strains were lower in
the case of the transplant of 1.times.10.sup.5 cells, and
particularly tumor proliferation was not observed in any of the
C.B-17/Icr-scid mice.
[0173] In the case of the transplant of 1.times.10.sup.6 cells, the
growth curves of the tumors are shown in FIG. 16.
[0174] The tumor proliferation in NOG mice exceeded that of the
other 2 strains.
[0175] On the 21st day, the NOG mice had 2.89 and 3.97 times larger
average tumor volume than the NOD/Shi-scid mice and C.B-17/Icr-scid
mice, respectively, had on the same day, and significant
differences were observed by Student t-test. (p<0.001)
[0176] No significant difference was observed in the average volume
on the 21st day between tumors engrafted in NOD/Shi-scid mice and
tumors engrafted in C.B-17/Icr-scid mice.
Example 6
Establishment of HIV-Infection Model System Using NOG Mice
[0177] Establishment of HIV-infection model system using NOG mice
was examined by use of various HIV lines. Typical examples are
shown below.
[0178] NOG mice were used in this example. For comparison,
NOD/Shi-scid mice were also used, which were produced before by
backcrossing both mutants of C.B-17-scid mice and NOD/Shi mice with
each other.
[0179] HIV-1 used herein was a JRFL virus which was isolated from
frontal lobe tissue of a patient with AIDS-associated
encephalopathy and had infectivity on a DNA-cloned macrophage and
T-cells. Further, a GFP-HIV-1 in which a JRFL virus and a GFP gene
were incorporated at env V3 region and downstream of gp41,
respectively was used.
[0180] (1) Production of Mice (hu-PBL-NOG Mice) to which Human
Mononuclear Lymphocytes are Transplanted
[0181] 1.times.10.sup.7 per mouse of peripheral blood human
lymphocytes (PBL), provided by a normal person with consent and
approval for test use, were intraperitoneally inoculated directly
into NOG mice. For comparison, the PBL from the same donor was
intraperitoneally inoculated into NOD/Shi-scid mice having a normal
IL-2R.gamma. chain.
[0182] (2) Infection of Mice with HIV-1
[0183] On the 6th day after the PBL inoculation, 1,000 TCID.sub.50
of viruses were intraperitoneally inoculated into mice. The mice
were sacrificed 2 weeks after the virus infection and ascites
including abdominal exudates cells were collected. The number of
human cells in the ascites was calculated by FACS. Further, spleens
were extracted and fixed by paraformaldehyde, and then made into
paraffin-embedded sections.
[0184] (3) Pathological Analysis
[0185] The spleen tissue sections were subjected to HE-stain, and
further to immunostaining using antibodies against human CD3, CD4,
CD8, and HIV-1p24 antigens. For immunohistologic stain, in addition
to conventionally reported ABC method, EPOS method and Envisiont
method were employed.
[0186] The engraftment of human cells in hu-PBL-NOG mice was
evaluated.
[0187] A series of examination results are shown in Table 5.
TABLE-US-00006 TABLE 5 HIV-1 infection test on human PBMC
grafted-NOG mice female without TM .beta.1 non existent PBL NOG
mice PBL transplantation total PBL 160 mL 4.37 .times. 10.sup.8
cells 2 .times. 10.sup.7 cells per mouse Donor A 14 NOG mice (6
males, 8 females) Infection NOG mice JRFL M-tropic 3 males NL4-3
T-tropic 3 males JRCSF M-tropic 3 females NLCSFV3EGFP M-tropic 2
females MOCK 3 females 13th day after infection Cell number
.times.10.sup.4 cells % HLA % CD4 % CD8 p24 1. Mock Ascites 75 67.7
21.4 56.4 Spleen 510 71.8 13.8 45.5 PBL 430 77.9 16.8 49.9 0 2.
Mock Ascites 130 67.6 14.8 60.5 Spleen 698 63.3 12.7 49.2 PBL 86
66.6 11.6 55.5 0 3. Mock Ascites 110 62.3 36.4 44.2 Spleen 434 61.3
20.9 45.2 PBL 93 34.7 20.6 44.7 0 HIV-1 infection to human PBMC
grafted-NOG mice (2) Cell number .times.10.sup.4 cells % HLA % CD4
% CD8 p24 4. JRFL Ascites 152 75.5 1.7 53.9 Spleen 335 54.6 2.8
64.6 PBL 210 42.8 2.1 77.3 3,090 5. JRFL Ascites 220 75.8 1.8 57
Spleen 12 75.8 1.65 79.6 PBL 870 81.1 1.8 77.9 5,445 6. JRFL
Ascites 178 36.3 1.79 54.1 Spleen 418 65.3 1.1 74.4 PBL 65 33.1
1.72 75.6 1,961 7. NL4-3 small spleen Ascites 120 7.6 9.74 74.1
Spleen 14 no FACS PBL non-existent 8. NL4-3 Ascites 84 75.5 1.43
45.7 Spleen 465 53 1.64 60.0 PBL 26 11.3 4.59 49.3 118 9. NL4-3
Ascites 110 8.1 9.33 80.6 Spleen 17 14.8 19.1 23.5 PBL 3 0.6 13.0
27.8 0 for three male NL4-3, their ears were excised. 10. JRCSF
Ascites 320 29.9 0.77 93.7 Spleen 230 67 1.85 74.9 PBL 49 33.7 0.48
71.7 3,132 11. JRCSF Ascites 63 68.5 3.18 41.6 Spleen 390 68.7 3.58
85.8 PBL 121 2.9 4.5 31.5 4,180 12. JRCSF poor construction Ascites
110 15.2 23.0 62.4 Spleen 380 14.5 2.98 89.8 PBL 110 44.7 2.06 89.4
864 13. NLCSFV3EFFP GFP+ of spleen and PBL are 0.1% or less Ascites
43 72.5 2.26 69.8 Spleen 250 50.8 0.77 67.7 PBL 124 76.5 5.07 63.3
271 14. NLCSFV3EGFP GFP+ of ascites, spleen and PBL are 0.2 to 0.3%
or less Ascites 110 44.3 6.36 76.3 Spleen 560 2.7 23.3 44.4 PBL 127
3.4 5.26 84.2 114
[0188] CD4 positive cells were specifically killed by all the HIV
viruses.
[0189] Conventionally, it has been reported that only M-tropic
viruses can proliferate in mice and CD4 positive cells are killed,
but T-tropic viruses can proliferate in NOG mice.
[0190] It was found that human CD4 positive T-cells and CD8
positive T-cells were each engrafted around central arteries of
spleens of the mice to which human peripheral blood was
transplanted. With respect to the number of human CD3 positive
cells, NOG mice had obviously a larger number of human cells than
NOD/Shi-scid mice. According to the measurement by FACS, there were
1.times.10.sup.7 or more human CD4 positive cells on average in
ascites.
[0191] On the other hand, CD4 positive cells were hardly detected
from NOD/Shi-scid mice and almost all the detected cells were CD8
positive cells. In the example previously described, the antibodies
(TM.beta.-1) (0.5 mg per mouse) which were against mouse IL-2
receptor .beta. chain and suppressed the differentiation of NK
cells were intraperitoneally injected into the NOD/Shi-scid mice 3
days before the inoculation of human PBL. However, this treatment
was not conducted in this example. It has been already confirmed in
other tests that antibody-treated mice would have many CD4 and CD8
positive human cells engrafted to them. Still though the number of
human cells from NOG mice was apparently 3 to 4 times more than the
number of cells from NOD/Shi-scid mice that had been subjected to
the antibody treatment.
[0192] In the HIV-infected mice, p24 positive cells which were HIV
antigens were detected around spleen arteries. Further, in spleens
and ascites of the HIV-infected mice which express GFP, cells with
GFP expression were clearly detected. The number of virus-infected
cells of NOG mice was apparently larger than that of NOD/Shi-scid
mice.
Example 7
Establishment of HTLV-1 Infection Model System Using NOG Mice
[0193] 1.times.10.sup.7 cells per mouse of MT-2 cells which were
cell lines derived from HTLV-1 leukemia, were transplanted to NOG
mice.
[0194] In the 4th week after the transplant, the mice having
lymphomas formed therein (Table 6) were counted, and it was checked
whether or not the mice having lymphomas were infected with a HTLV
gene, by Southern blotting and PCR. As a result of a pathological
search, dermal lymphoma and posterior intraperitoneally bilateral
lymphomas are observed. The mice having a large tumor had
metastasis in lymph nodes around the stomach and had cancer cells
infiltrated in a pleura thereof. Further, HTLV positive cells
increased in peripheral blood. This indicated a typical onset of
leukemia. Moreover, lung-interstitial invasion was also observed.
All of these cells expressed Tax protein, but there was no
intracerebral invasion.
TABLE-US-00007 TABLE 6 1. Transplantation of HTLV-infected cells to
NOG mice 2 .times. 10.sup.7 cells per mouse Cell No. of mice
treatment having lymphoma No. by (after 1 of mice TM.beta.-1 gamma
ray month) NOD/Shi-scid mice 5 -- -- 0 NOG mice 40 -- -- 33 (82.5%)
NOG mice 4 -- 20,000 rad 0 NOG mice (no cell 2 -- -- 0
transplanted)
Example 8
Examination of Leukemic Changes by Introducing Neoplastic Cells and
Transplantability of HTLV-1 Positive Cells Using NOG Mice
[0195] Transplant tests of various HTLV-1 cell lines and tumor
formation tests were performed on NOG mice and the usefulness of
the mice was examined.
[0196] Using 3 samples of HTLV-1 leukemia derived cell lines
[L=Leukemic cell lines: ED-40515(-), MT-1 and TL-Oml], 6 samples of
HTLV-1 infected cell lines [IT=infected transformed cell lines:
SLB-1, M8116, HUT-102, MT-2, MT-4 and TY9-31MT], 10.sup.7-8
cells/0.5 mL were transplanted to NOG mice and NOD/Shi-scid mice
subcutaneously at a left posterior auricle thereof, and to some of
them subcutaneously at a gluteal region thereof. Then, the tumor
size in the mice, tissue image, and the mice with, and those
without, leukemia were chronologically compared for examination,
and the relations between these and transcription factors such as
NFkB or fluctuation of Tax protein were also analyzed.
[0197] Within 2 weeks, a tumor with a size of 24.times.17.times.11
mm was formed in all cases of HTLV-1 leukemia derived cell lines
[L] and exceptionally in SLB-1 of HTLV-1 infected cell line [IT].
In contrast, in 5 out of 6 cases of IT, a tumor with a size of at
most 10.times.10.times.7 mm was formed, and in the case of M8116 or
TY9-31MT, there was almost no tumor formation. (Table 7)
TABLE-US-00008 TABLE 7 Development of HTLV-1 infected-cells and
leukemia cells in NOG mice and their features in vitro and in vivo
In vitro No. of proliferation II-2 Cell inoculated Route and site
of In vivo Cell line Cell type mice pattern dependence Tax
expression (.times.10.sup.7) inoculation period (days) size of
tumor (mm) ED-40516(-) l 1 separately - - 7.5 I.m Buttock 16 23
.times. 18 .times. 12 7 separately - - 7.5 s.c post-auricular 15-16
25 .times. 18 .times. 12 3 separately - - 4 s.c post-auricular 16
25 .times. 18 .times. 12 3 separately - - 1 s.c post -auricular 15
22 .times. 17 .times. 10 SLB-1 IT 1 clustered - + 7.5 I.m Buttock
15 20 .times. 10 .times. 18 7 clustered - + 7.5 s.c post-auricular
15-16 20 .times. 10 .times. 18 3 clustered - + 5 s.c post-auricular
15 21 .times. 12 .times. 17 MT-1 L 1 separately - - 7.5 I.m Buttock
20 25 .times. 15 .times. 10 1 separately - - 7.5 s.c post-auricular
20 25 .times. 15 .times. 10 3 separately - - 4 s.c post-auricular
24 23 .times. 16 .times. 12 TI-om1 L 1 separately - - 7.5 I.m
Buttock 20 25 .times. 15 .times. 10 1 separately - - 7.5 s.c
post-auricular 20 25 .times. 15 .times. 10 3 separately - - 4 s.c
post-auricular 20 22 .times. 23 .times. 8 M8116 IT 1 clustered - +
7.5 I.m Buttock 18 sesame 2 clustered - + 7.5 s.c post-auricular 18
sesame HUT-102 IT 1 clustered - + 7.5 I.m Buttock 18 10 .times. 10
.times. 7 2 clustered - + 7.5 s.c post-auricular 18 10 .times. 10
.times. 7 MT-2 IT 1 clustered - + 7.5 I.m Buttock 18 10 .times. 10
.times. 7 2 clustered - + 7.5 s.c post-auricular 18 10 .times. 10
.times. 7 MT-4 IT 1 clustered - + 7.5 I.m Buttock 18 5 .times. 5
.times. 3 2 clustered - + 7.5 s.c post-auricular 18 5 .times. 5
.times. 3 TY9-31MT-2 IT 1 clustered - + 7.5 I.m Buttock 18 sesame 2
clustered - + 7.5 s.c post-auricular 18 sesame L: leukemia cell
line IT: infected-tranformant cell line
[0198] [ ] The case of this tumorigenic cell line showed that the
degree of the emergence of leukemic cells in peripheral blood and
the invasion of organs were relatively high, but some cases of
nontumorigenic cell lines had higher degrees thereof (e.g. HUT-102,
MT-2 etc.). Thus, there were not necessarily correlations between
tumorigenicity and the degrees of the emergence of leukemic cells
in peripheral blood and the invasion on organs. (Table 8).
TABLE-US-00009 TABLE 8 Infiltration of leukemia cells into various
organs of NOG mice bone peripheral marrow Cell strain Tumor blood
(PB) (BM) liver spleen lung brain kidney heart Ed-40515(-) +++ ++ -
+ + +- Spongy+- ++ - SLB-1 +++ +++ + ++ + Granular+++ Spongy++ + +
MT-1 +++ ++ - + + + Spongy+- - - TI-oml +++ ++ - Giant+++ + +- - -
- M8116 + + - - - - - - - Hut-102 ++ ++ +++ Nodule+++ +++
Pneumonia+ Spongy+- - - MT-2 ++ ++ +- +- ++ +- - + - MT-4 ++ + +- -
++ - - - - TY9-31MT-2 +- - - - - - - - -
[0199] [ ] 10.sup.7 cells of tumorigenic ED-40515(-) were
transplanted subcutaneously at a left posterior auricle of NOG mice
and NOD/Shi-scid mice and they were compared in tumorigenicity in
the second week. A tumor with a size of 22.times.17.times.10 mm was
formed in the NOG mice, and in contrast a tumor was hardly formed
at all in NOD/Shi-scid mice. This revealed that NOG mice were prone
to tumor formation. (Table 9)
TABLE-US-00010 TABLE 9 Comparison of tumor formation between NOG
mice and NOD/Shi-scid mice to which ED-40515(-) cell type was
transplanted Cells Survival Mouse strain No. of mice inoculated
(.times.10.sup.7) period (days) Tumor NOG mouse 3 1 15 +++
NOD/Shi-scid 3 1 15 - mouse +++ tumor large enough to visually
observe - tumor which cannot visually be observed
[0200] For the purpose of investigating whether or not the NOG mice
are prone to tumor formation in B-cell type, as well as in T-cell
type, 7.times.10.sup.7 of BJAB cells of which only EBER
(EBV-Encoded Small RNA) and a vector were transformed, were
transplanted subcutaneously at a left posterior auricle of NOG
mice. In the 3rd week, they were compared in tumorigenicity. A
significantly large tumor (26.times.18.times.7/13.times.18.times.3
mm) was formed in the mice with BJAB-EBER compared to the mice with
BJAB-VECTOR. The tumor size thereof was comparable to that in the
case of ED-40515(-) in the above example. (Table 10)
TABLE-US-00011 TABLE 10 Proliferation of BJAB-EBER cell line and
BJAB-VECTOR cell line in NOG mice No. Cells Survival Weight Gene of
inoculated Route and site of period Tumor of Cell line introduction
Mice (.times.10.sup.7) inoculation (days) size (mm) tumor BJAB-EBER
gene 3 7 s.c. post-auricilar 21 26 .times. 18 .times. 7 2.86 g
introduced BJAB-VECTOR gene not 3 7 s.c. post-auricular 21 13
.times. 18 .times. 3 0.73 g introduced
[0201] Assuming that effective tumor formation by 10.sup.7 of
ED-40515(-) in NOG mice was attributable to active neogenesis of
tumoral vessels via CXCR4 of tumor cells, it has been expected that
tumor formation would be inhibited as long as there was continuous
administration of KRH-1636, a competitive agent of SDF-1 which was
a ligand thereof in endothelial cells. However, in the case of
ED-40515(-) and SLB-1 (histologically angiotropic) accompanied by
bleeding, the mice formed tumors with sizes of 25.times.18.times.12
mm and 20.times.10.times.18 mm equivalent to those of non-treated
group, even though KRH-1636 was intraperitoneally administered
every day.
TABLE-US-00012 TABLE 11 Effect of CXDR4 antagonists on In Vivo
Growth and Proliferation of HTLV-1 infected cell lines in NOG mice
Cells No. of inoculated Route of drug Survival Tumor size Cell line
group mice (.times.107) administration period (days) (mm) Remarks
ED-40515(-) Drug 3 7.5 intraperitoneally 15 25 .times. 18 .times.
12 progressive large tumor KRH-1636 0.14 mg/mouse Control 3 7.5
intraperitoneally 15 25 .times. 18 .times. 12 progressive large
tumor medium 0.2 ml/mouse SLB-1 Drug 3 7.5 intraperitoneally 15 20
.times. 10 .times. 18 progressive large tumor KRH-1636 with
hemorrhage 0.14 mg/mouse Control 3 7.5 intraperitoneally 15 20
.times. 10 .times. 18 progressive large tumor medium with
hemorrhage 0.2 ml/mouse
[0202] [ ] Using Cytospin specimens of in vitro culture cells and
frozen section specimens of in vivo tumor formative cells, both
from ED-40515(-) and SLB-1, the immunostaining manners of CD4, CD8,
CD3, CXCR4, CCR5 and SDF-1 were comparatively tested by enzyme
antibody technique. All the cases of CD4, CD3, CXCR4 and SDF-1 were
almost equally positive, and all the cases of CCR5 were negative.
CD8 was negative in vitro, but positive in vivo. (Table 12)
TABLE-US-00013 TABLE 12 In vitro and in vivo examination of HTLV-1
infected cell line-transplanted NOG mice by FACS, WB, EMSA and
immunohistochemistry Immunohistochemistry CD4 CD8 CD3 CXCR4 CCR5
SDF-1 In-vitro ED-40515(-) ++ - + + - ++ SLB-1 ++ - + + - ++
In-vivo ED-40515(-) +++ + + + - +++ SLB-1 +++ + + + - +++ +++
strongly positive ++ positive + weakly positive - negative
[0203] Using in vitro culture cells and in vivo tumor formative
cells of ED-40515(-), Table 13 Western blotting analyses on Tax,
CXCR4, OX40 and OX40L were performed. All the cases of Tax were
negative. However, all the cases of CXCR4, OX40 and OX40L were
positive, and there was no significant difference in their
strengths. (Table 13)
In Vitro and In Vivo Examination of HTLV-1 Infected Cell
Line-Transplanted NOG Mice by FACS, WB, EMSA and
Immunohistochemistry
TABLE-US-00014 [0204] In-vitro WB TAX CXCR4 OX40 OX40L ED-40515(-)
- ++ + + SLB-1 ++ ++ +++ -
TABLE-US-00015 In-vivo TAX CXCR4 OX40 OX40L - + + +
[0205] Using in vitro culture cells and in vivo tumor formative
cells of ED-40515(-), transcription factor activity of NFkB was
examined by electrophoretic mobility shift assay (EMSA), though no
difference therebetween was observed. (Table 14)
TABLE-US-00016 TABLE 14 In vitro and in vivo examination of HTLV-1
infected cell line-transplanted NOG mice by FACS, WB, EMSA and
immunohistochemistry In-vitro In-vivo EMSA NFkB NFkB ED-40515(-)
+++ +++ SLB-1 +++
[0206] The following points were recognized in the system wherein
the transplant was conducted subcutaneously at posterior auricles
of the NOG mice.
[0207] (1) It was certainly confirmed that large tumors were formed
in the NOG mice within very short periods, like 15 days after
inoculation, which had not been expected. Conversely, NOD/Shi-scid
mice (IL-2R .gamma. chain.sup.+/+) did not form any tumors.
[0208] (2) It was revealed that the NOG mice genetically delete NK
cells, and therefore they did not require the pre-treatment against
anti NK cells using monoclonal antibodies etc., which is essential
in the case of C.B-17/Icr-scid or NOD/Shi-scid mice. (IL-2R .gamma.
chain.sup.+/+)
[0209] (3) The transplant was performed subcutaneously at a
posterior auricle, that is, selecting a site which has a
anatomically lower number of NK cells than an intraperitoneal site,
and this allows a tumor to be easily formed. As a result, the size
of the tumor was easily observed by appearance without need for
incision.
[0210] (4) Although Uchiyama et al. reported that MT-1, T-2 and
TL-Oml did not form any tumors, in the present case relatively
small tumors were formed in them. (Imada K, Takaori-Kondo A, Akagi
T, Shimotohno K, Sugamura K, Hattori T, Yamabe H, Okuma M, Uchiyama
T: Tumorigenicity of human T-cell leukemia virus type I-infected
cell lines in severe combined immunodeficient mice and
characterization of the cells proliferating in vivo. Blood
86:2350-7, 1995, Uchiyama, T: Human T cell leukemia virus type I
(HTLV-I) and human diseases. Annu Rev Immunol 15:15-37, 1997).
[0211] Therefore, the system wherein the transplant is carried out
subcutaneously at posterior auricles of the NOG mice, is an
innovative tumor transplant system. Further, it was revealed that
it could work in the same manner on B-cell tumors as well as on
T-cell type tumors. Namely, these indicate also that this system is
a valuable transplant system for the transplant of cancer cells or
human normal lymphoid tissues.
[0212] In addition, when there is no laboratory animal model or it
is difficult, even if there is, to put one into practical use, a
human disease model can be established by transplanting human cells
or tissues to this NOG mouse, thereby enabling disease researches,
for which there has been no other choice but to be dependent on in
vitro tests, with a test system which comes close to a limitless
human in vivo test. Thus, this mouse is considered to make great
contributions to the elucidation of mechanisms of disease onset,
development of therapies etc. For example, irrespective of potent
HAART therapy against HIV-1 infection, the rebound or mutant virus
emergence of viremia is of primary importance. The reservoir of
infectious viruses thereof is known to be FDC (follicular dendritic
cells) of lymphatic follicle, and for researches on it, it is
indispensable to establish a humanized model mouse to which
lymphoid tissues including human lymphatic follicle are
transplanted. For this, the present NOG mouse is useful.
[0213] Furthermore, with respect to a cause to make this system
prone to form a tumor, examinations were made on what factor is
increased or activated in transplanted cells, as compared with in
vitro culture cells. All the cell lines used in the above examples
were IL-2 independent, IL-2 producing, further Tax and CXCR4-SDF-1
system were revealed not to be directly associated therewith.
However, within the search the present inventors have made, there
was no factor except the above mentioned which the inventors
recognized indicated a significant difference in comparison between
in vitro culture cells and in vivo tumor-forming cells.
INDUSTRIAL APPLICABILITY
[0214] The present invention provides a method of producing NOG
mice more suitable for engraftment of heterologous cells,
particularly human cells, and a mouse produced by the method, as
compared with an NOD/Shi-scid mouse and an NOD/LtSz-scid, .beta.2m
null mouse both conventionally known as an immunodeficient mouse.
Transplanting human stem cells to the thus obtained mice enables a
human stem cell assay system to be established. Further,
transplanting human cells responsible for immunity enables human
antibodies to be produced using the mouse of the present invention.
Furthermore, a human tumor model mouse can be produced by
transplanting and engrafting human tumors to the mouse, and the
mouse can be used for therapies for tumors, screening of
therapeutic agents and the like. Moreover, it is possible to
produce an HIV or HTLV-1 infected model mouse to which an HIV or
HTLV1 infected human lymphocyte is engrafted, and researches can be
made on in vivo proliferation mechanism of HIV-1, HTLV-1 etc. Also,
it is possible to conduct development of a therapy for virus
infection, screening of a therapeutic agent for virus infection or
the like.
[0215] All publications cited herein are incorporated herein with
the whole contents thereof. Further, it is easily understood by
those skilled in the art that various changes and modifications can
be made in the invention without departing from the technical idea
and scope described in the appended claims. It is intended that the
present invention cover all such changes and modifications.
Sequence CWU 1
1
3126DNAArtificial SequencePrimer PI 1ctgctcagaa tgatgcctcc aattcc
26226DNAArtificial SequencePrimer PII 2cctgcgtgca atccatcttg ttcaat
26324DNAArtificial SequencePrimer PIII 3gatccagatt gccaaggtga gtag
24
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