U.S. patent application number 10/493526 was filed with the patent office on 2005-02-17 for sgrf gene-modified non-human animals.
Invention is credited to Habu, Kiyoshi, Hirata, Yuichi.
Application Number | 20050039222 10/493526 |
Document ID | / |
Family ID | 19143169 |
Filed Date | 2005-02-17 |
United States Patent
Application |
20050039222 |
Kind Code |
A1 |
Habu, Kiyoshi ; et
al. |
February 17, 2005 |
Sgrf gene-modified non-human animals
Abstract
A targeting vector was constructed by replacing exon regions in
the SGRF gene with appropriate drug marker genes. This vector was
transfected into mouse ES cell lines to obtain chimeric mice, which
were then crossed with C57BL/6J mice to obtain mice comprising
cells in which one SGRF gene alleles was inactivated. By crossing
these mice with each other, the present inventors succeeded in
producing mice in which both SGRF gene alleles were inactivated.
These genetically modified animals can be used to predict the side
effects of drugs such as SGRF antagonists.
Inventors: |
Habu, Kiyoshi; (Shizuoka,,
JP) ; Hirata, Yuichi; (Ibaraki, JP) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Family ID: |
19143169 |
Appl. No.: |
10/493526 |
Filed: |
October 6, 2004 |
PCT Filed: |
October 24, 2002 |
PCT NO: |
PCT/JP02/11047 |
Current U.S.
Class: |
800/18 ;
424/145.1; 435/354; 435/7.2 |
Current CPC
Class: |
A61P 17/00 20180101;
A61P 11/06 20180101; A61P 15/00 20180101; A61P 19/02 20180101; A01K
2267/03 20130101; A61P 9/10 20180101; A61P 13/12 20180101; C12N
2800/30 20130101; A01K 67/0276 20130101; A61P 9/00 20180101; A61P
37/00 20180101; A61P 29/00 20180101; A61P 5/14 20180101; A61P 1/04
20180101; A01K 67/0271 20130101; A61P 27/02 20180101; A61P 15/06
20180101; A61P 37/08 20180101; A61P 21/04 20180101; A01K 2217/075
20130101; A61P 7/06 20180101; A61P 37/02 20180101; A61P 1/16
20180101; A61P 11/00 20180101; A61P 21/00 20180101; C12N 15/8509
20130101; A61P 7/00 20180101; A61P 17/02 20180101; A01K 2227/105
20130101; A61P 3/10 20180101; A61P 31/00 20180101; A01K 2267/0368
20130101; A61P 17/06 20180101; A61P 19/06 20180101; A61P 25/28
20180101; A01K 2267/0325 20130101; A61P 1/02 20180101; A61P 25/00
20180101 |
Class at
Publication: |
800/018 ;
435/354; 424/145.1; 435/007.2 |
International
Class: |
A01K 067/027; A61K
039/395; C12N 005/06; G01N 033/53; G01N 033/567 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 24, 2001 |
JP |
2001-326858 |
Claims
1. A genetically modified non-human animal in which SGRF gene
expression is artificially suppressed.
2. A genetically modified non-human animal in which a foreign gene
is inserted into one or both of the SGRF gene alleles.
3. The genetically modified animal of claim 1, in which the
non-human animal is a rodent.
4. The genetically modified animal of claim 3, in which the rodent
is a mouse.
5. A cell line established from a genetically modified animal of
claim 1.
6. A method for producing an anti-SGRF antibody, wherein the method
comprises using a genetically modified animal of claim 1.
7. A non-human ES cell in which SGRF gene expression is
artificially suppressed.
8. A non-human ES cell in which a foreign gene is inserted into one
or both of the SGRF gene alleles.
9. The ES cell of claim 7, in which the non-human ES cell is a
rodent ES cell.
10. The ES cell of claim 9, in which the rodent ES cell is a mouse
ES cell.
11. An infection-preventing agent, comprising a compound that can
substitute for SGRF or a SGRF signal, as its active ingredient.
12. A therapeutic agent for autoimmune diseases, which comprises an
SGRF antagonist as its active ingredient.
13. A therapeutic agent for inflammatory diseases, which comprises
an SGRF antagonist as its active ingredient.
14. The therapeutic agent of claim 12, in which the antagonist is
an anti-SGRF antibody.
15. A method of screening for a compound as a candidate for a
therapeutic agent for autoimmune or inflammatory diseases, wherein
the method comprises the steps of (a) to (c): (a) contacting SGRF
with a test compound; (b) measuring the binding activity of SGRF
with a test compound; (c) selecting a compound which binds to
SGRF.
16. A method of screening for a compound which can substitute for
SGRF protein function, wherein the method comprises the steps of
(a) to (c): (a) administering a test compound into a genetically
modified animal of claim 1; (b) determining whether the test
compound can substitute for SGRF function; (c) selecting a compound
that can substitute for SGRF function.
17. The genetically modified animal of claim 2, in which the
non-human animal is a rodent.
18. The genetically modified animal of claim 17, in which the
rodent is a mouse.
19. A cell line established from a genetically modified animal of
claim 2.
20. A method for producing an anti-SGRF antibody, wherein the
method comprises using a genetically modified animal of claim
2.
21. The ES cell of claim 8, in which the non-human ES cell is a
rodent ES cell.
22. The ES cell of claim 21, in which the rodent ES cell is a mouse
ES cell.
23. The therapeutic agent of claim 13, in which the antagonist is
an anti-SGRF antibody.
24. A method of screening for a compound which can substitute for
SGRF protein function, wherein the method comprises the steps of
(a) to (c): (a) administering a test compound into a genetically
modified animal of claim 2; (b) determining whether the test
compound can substitute for SGRF function; (c) selecting a compound
that can substitute for SGRF function.
Description
TECHNICAL FIELD
[0001] The present invention relates to SGRF gene knockout
non-human animals and cells, and to uses thereof.
BACKGROUND ART
[0002] SGRF (Interleukin-Six, G-CSF Related Factor)/IL-23
(hereafter indicated as "SGRF") is a novel cytokine cloned by the
present inventors based on an EST (Patent number EP1072610, GenBank
Accession No. AB030000). Oppmann et al. also isolated this gene
using similar methods, reporting it as a novel cytokine (p19/IL-23)
that exerts its physiological function by combining with the p40
subunit of IL-12 (Immunity Vol. 13, p715, 2000). However, to date
the physiological function of this gene has been unclear, except
for the following: (1) it induces proliferation of
CD4.sup.+CD45RB.sup.low T cells in mice, and (2) it induces
IFN-.gamma. production in human memory T cells. Analysis using
IL-12 p40 deficient mice (knockout mice) and research using an
antibody against IL-12 p40 has introduced the possibility that
functions conventionally attributed to IL-12 could in fact be due
to SGRF function.
[0003] The present inventors sought effective experimental systems
for use in elucidating the functions of the SGRF gene, which were
as yet unknown. Genetically modified animals in which SGRF gene
allele inactivation lead to lack of SGRF enzymatic activity, or
cell lines derived from these genetically modified animals
(genetically modified mouse-derived cell lines) or ES cell lines
can be utilized to elucidate the physiological function of SGRF at
both the cellular and whole individual levels. Thus, such animals
and cell lines can be used to develop therapeutic drugs to treat
diseases associated with the SGRF gene.
DISCLOSURE OF THE INVENTION
[0004] The present invention was carried out under such
circumstances. An object of the present invention is to provide
genetically modified animals in which both SGRF gene alleles have
been inactivated, and therefore to provide animals which do not
possess SGRF activity. Another object of the present invention is
to provide cell lines derived from these animals, ES cells in which
expression of the SGRF gene has been suppressed, as well as
screening methods for compounds that substitute for SGRF function
in these animals. Another object of the present invention is to
provide therapeutic drugs for diseases associated with SGRF, based
on observations of SGRF function obtained using these genetically
modified mice.
[0005] The present inventors generated SGRF gene-modified animals
in order to achieve the above-described objectives. SGRF gene exon
region was replaced with appropriate drug marker genes, thereby
generating a targeting vector. This targeting vector was
transfected into mouse-derived ES cell lines, and then cell lines
in which homologous recombination had occurred were selected. ES
cells in which one of the SGRF gene alleles was inactivated were
injected into a C57BL/6J mouse-derived blastocyst, and chimeric
mice were obtained. Mice in which one of the SGRF gene alleles had
been inactivated (SGRF/IL-23.sup.+/-) were obtained by crossing
these chimeric mice with C57BL/6J mice. Mice in which both SGRF
gene alleles were inactivated (SGRF/IL-23.sup.-/-) were
successfully obtained by crossing SGRF/IL-23.sup.+/- mice with each
other.
[0006] Such genetically modified animals and ES cells can be used
to predict the side effects of SGRF inhibitors, including, for
example, anti-SGRF antibodies or SGRF antagonists. In addition, the
genetically modified animals and ES cells of this invention can be
used to examine whether or not test proteins and small molecules
have the ability to substitute for SGRF function, and also to
screen for DNAs that encode proteins comprising the ability to
substitute for SGRF function.
[0007] The present inventors used the above-described genetically
modified animals to reveal the association of SGRF's physiological
functions with disease. For example, the genetically modified mice
were found to be susceptible to infection by the pathogenic
microbe, Listeria. Therefore, SGRF may be a therapeutic drug for
pathogenic microbes. The induction of experimental autoimmune
encephalomyelitis (EAE) does not easily occur in genetically
modified mice, and had a low delayed-type hypersensitivity
response. Therefore, it is suggested that SGRF is associated with
the development of autoimmune and inflammatory diseases in humans.
Hence, compounds that inhibit SGRF function are expected to be
drugs for treating autoimmune or inflammatory diseases.
[0008] SGRF is expressed in the brain, and SGRF/IL-23.sup.-/- mice
do not easily develop EAE, indicating involvement of SGRF in
encephalopathy. Therefore, compounds that inhibit SGRF function are
expected to be drugs for treating encephalopathy.
[0009] Thus, the present invention relates to genetically modified
animals in which SGRF gene allele inactivation has eliminated SGRF
enzymatic activity, cell lines derived from these animals, and ES
cells in which expression of the SGRF gene is suppressed. The
present invention also relates to uses for screening compounds that
can substitute for SGRF function using these animals, and to
therapeutic drugs for diseases associated with SGRF. More
specifically, the present invention provides:
[0010] (1) a genetically modified non-human animal in which SGRF
gene expression is artificially suppressed;
[0011] (2) a genetically modified non-human animal in which a
foreign gene is inserted into one or both of the SGRF gene
alleles;
[0012] (3) the genetically modified animal of (1) or (2), in which
the non-human animal is a rodent;
[0013] (4) the genetically modified animal of (3), in which the
rodent is a mouse;
[0014] (5) a cell line established from a genetically modified
animal of any one of (1) to (4);
[0015] (6) a method for producing an anti-SGRF antibody, wherein
the method comprises using a genetically modified animal of any one
of (1) to (4);
[0016] (7) a non-human ES cell in which SGRF gene expression is
artificially suppressed;
[0017] (8) a not-human ES cell in which a foreign gene is inserted
into one or both of the SGRF gene alleles;
[0018] (9) the ES cell of (7) or (8), in which the non-human ES
cell is a rodent ES cell;
[0019] (10) the ES cell of (9), in which the rodent ES cell is a
mouse ES cell;
[0020] (11) an infection-preventing agent, comprising a compound
that can substitute for SGRF or a SGRF signal, as its active
ingredient;
[0021] (12) a therapeutic agent for autoimmune diseases, which
comprises an SGRF antagonist as its active ingredient;
[0022] (13) a therapeutic agent for inflammatory diseases, which
comprises an SGRF antagonist as its active ingredient;
[0023] (14) the therapeutic agent of (12) or (13), in which the
antagonist is an anti-SGRF antibody;
[0024] (15) a method of screening for a compound as a candidate for
a therapeutic agent for autoimmune or inflammatory diseases,
wherein the method comprises the steps of (a) to (c):
[0025] (a) contacting SGRF with a test compound;
[0026] (b) measuring the binding activity of SGRF with a test
compound;
[0027] (c) selecting a compound which binds to SGRF;
[0028] (16) a method of screening for a compound which can
substitute for SGRF protein function, wherein the method comprises
the steps of (a) to (c):
[0029] (a) administering a test compound into a genetically
modified animal of any one of (1) to (4);
[0030] (b) determining whether the test compound can substitute for
SGRF function;
[0031] (c) selecting a compound that can substitute for SGRF
function.
[0032] The present invention provides genetically modified
non-human animals and ES cells characterized by artificial
suppression of SGRF gene expression.
[0033] The SGRF (Interleukin-Six, G-CSF Related Factor) gene (the
protein encoded by this gene is described as "SGRF") of the present
invention has been identified in, for example, humans (GenBank
Accession No. AB030000) and mice (GenBank Accession No. AF301619).
The SGRF gene consists of four exons (GenBank Accession No.
AB030001). In the present invention, "SGRF gene expression is
artificially suppressed" means that the expression of the SGRF gene
is suppressed due to genetic mutation, including insertion,
deletion, or substitution of nucleotides into one or both of the
SGRF gene alleles. Cases where the function of the normal SGRF
protein is reduced or lost due to the expression of a mutant SGRF
protein are also included in "suppression of SGRF gene expression".
"Suppression" as described above includes both cases where SGRF
gene expression is completely suppressed, and where expression of
only one SGRF gene allele is suppressed. In the present invention,
specific suppression of SGRF gene expression is preferred. There is
no restriction as to the sites into which mutations can be
introduced, as long as gene expression is suppressed. For example,
mutations can be introduced into exons or promoter regions.
[0034] Animals used for SGRF gene modification in the present
invention are usually animals other than humans, preferably rodents
such as mice, rats, hamsters and rabbits, and more preferably mice.
ES cells used for SGRF gene modification in the present invention
are preferably derived from rodents, and more preferably from mice.
Furthermore, animals generally referred to as "knockout animals"
are also included as genetically modified animals of the present
invention.
[0035] In the present invention, methods for artificially
suppressing the SGRF gene in genetically modified ES cells and
genetically modified non-human animals (also referred to as
"genetically modified animals") include deletion of part of or the
entire SGRF gene, or deletion of part of or the entire SGRF gene
expression regulatory region. Inactivation of the SGRF gene by the
insertion of a foreign gene into one or both of the SGRF gene
alleles is preferable. Therefore, in a preferred embodiment of the
present invention, a genetically modified animal or genetically
modified ES cell is characterized by the insertion of a foreign
gene into one or both of the SGRF gene alleles.
[0036] The genetically modified animal of the present invention can
be constructed using genetic engineering techniques well known to
those skilled in the art. For example, a genetically modified mouse
can be constructed as follows: First, DNA including the exons of
the SGRF gene is isolated from a mouse. A targeting vector is then
constructed by inserting an appropriate marker gene into this DNA
fragment. This targeting vector is introduced into a mouse ES cell
line using electroporation or the like, and a cell line in which
homologous recombination has occurred is selected. The marker gene
to be inserted is preferably a gene that is resistant to an
antibiotic, such as the neomycin-resistant gene. When such an
antibiotic-resistant gene is inserted, a cell line in which
homologous recombination has occurred can be easily selected by
simply incubating the cells in a medium containing the antibiotic.
For a more effective selection, a gene such as the thymidine kinase
gene can also be inserted into the targeting vector. This procedure
allows exclusion of cell lines in which non-homologous
recombination has occurred. Moreover, a cell line in which one of
the SGRF gene alleles has been inactivated can be efficiently
obtained by selecting homologous recombinants using PCR and
Southern blotting.
[0037] When cell lines produced by homologous recombination are
selected, chimeras are preferably constructed using multiple
clones, because gene insertion at sites other than homologous
recombination sites may have destroyed unknown genes. Chimeric mice
can be produced by injecting the obtained ES cell line into mice
blastocysts. Mice in which one SGRF allele is inactivated can be
obtained by crossing these chimeric mice with wild type mice. By
crossing the offspring of these crosses with each other, mice can
be obtained in which both SGRF alleles have been inactivated. More
specifically, it is possible to construct a genetically modified
mouse of the present invention according to the method described
below in the Examples. In addition to mice, it is also possible to
genetically modify other animals in which ES cells have been
established.
[0038] An ES cell line in which both SGRF alleles are inactivated
can be created by incubating an ES cell line in which one locus is
inactivated in a selective medium comprising a high concentration
of an antibiotic, upon which the other locus is inactivated. The
above-mentioned ES cell line can also be constructed by selecting
an ES cell line in which one locus is inactivated, reintroducing
the targeting vector into this cell line, and selecting a cell line
in which homologous recombination has occurred. The marker gene to
be inserted into the targeting vector is preferably different to
that used previously.
[0039] The present invention also provides cell lines established
from the genetically modified non-human animals of the present
invention. Cell lines can be established from the genetically
modified animals of the present invention using conventional
methods. For example, a primary culture method for embryonic cells
can be used in the case of rodents (Shin-seikagaku Jikken Kouza,
vol.18, pp 125-129, Tokyo Kagaku Doujin; and "Manuals for
manipulating mouse embryos", pp 262-264, Kindai Shuppan).
[0040] The genetically modified animals, cell lines established
from these animals, and ES cell lines of the present invention can
be used for detailed functional analysis of SGRF genes. For
example, the genetically modified animals, cell lines established
from these animals, and ES cell lines of the present invention can
be used for predicting the side effects of SGRF inhibitors such as
anti-SGRF antibodies or SGRF antagonist small molecules. SGRF
inhibitors (antagonists) are not considered to have lethal side
effects since the genetically modified mice obtained from the
present invention grew normally and did not die, at least during
fetal development. SGRF inhibitor side effects may also be
predicted by detailed examination of the genetically modified
animals of the present invention. SGRF inhibitor side effects in
each tissue can be precisely examined by using cell lines
established from tissues of the genetically modified animals.
Moreover, genetically modified animals of the present invention can
be used to screen for compounds that can substitute for the
function of the SGRF protein, as described below.
[0041] In the genetically modified animals of the present
invention, the SGRF gene is inactivated from birth, and thus the
animal can efficiently produce antibodies against SGRF-binding
proteins such as SGRF or IL-12 p40. For example, it is possible to
efficiently produce monoclonal or polyclonal antibodies against
SGRF by immunizing a genetically modified mouse of the present
invention with SGRF together with Freund's complete adjuvant. In
this case, the SGRF used for immunization can be derived from a rat
or a human, as well as a mouse.
[0042] Observation of the symptoms of the genetically modified
animals of this invention, and comparison of these symptoms with
the symptoms of diseases of unknown cause, will reveal if a disease
is caused by SGRF malfunction. For example, phenotypes
characteristically appearing in genetically modified mice or cell
lines derived from these mice, can be compared with various
symptoms of human disease. If more than half of the symptoms of a
human disease are observed in the genetically modified mice of this
invention, it can be presumed that the disease may be due to SGRF
malfunction.
[0043] As described in the Examples below, the present inventors
revealed SGRF's physiological function and association with disease
by generating and analyzing mice with an inactivated SGRF gene
(SGRF KO mice). These SGRF KO mice are susceptible to infection
with the pathogenic microbe Listeria. Therefore, SGRF and
alternative substances involved in SGRF signaling are expected to
be drugs against pathogenic microbes. Thus, the present invention
provides infection-preventing drugs that comprise SGRF as an active
ingredient.
[0044] Induction of experimental autoimmune encephalomyelitis (EAE)
is difficult in SGRF KO mice. SGRF is suspected to be an important
factor in the development of human autimmune diseases, including
multiple sclerosis (MS), and can be a target for treating such
autoimmune diseases. Thus, SGRF antagonists are expected to be
drugs for treating autoimmune diseases. Therefore, the present
invention provides therapeutic drugs for autoimmune diseases which
comprise an SGRF antagonist as an active ingredient.
[0045] SGRF KO mice have reduced delayed-type hypersensitivity, as
described in the Examples. Thus, suppression of SGRF function can
be a target for treating various inflammatory diseases, such as
asthma and allergies. Therefore, the present invention also
provides therapeutic drugs for inflammatory diseases which comprise
an SGRF antagonist as an active ingredient.
[0046] The present invention also provides therapeutic drugs for
encephalopathy which comprise an SGRF antagonist as an active
ingredient.
[0047] SGRF antagonists of the present invention include, for
example, anti-SGRF antibodies and anti-IL-12 p40 antibodies. SGRF
antagonists can be screened, for example, via the following
method:
[0048] Ba/F3 cells are known to react with IL-23, causing
proliferation when IL-12R.beta.1 and IL-23R are transfected into
Ba/F3 cells (J. Immunol., Vol.168, p.5699-5708, (2002)). Therefore,
a test compound that specifically inhibits IL-23 dependent
proliferation of such cell strains can be isolated as an
antagonist.
[0049] STAT4 phosphorylation is known to occur on addition of SGRF
to PHA-activated human premature T cells. In this system, compounds
which suppress STAT4 phosphorylation in the presence of SGRF, are
considered antagonist candidates. Therefore, SGRF antagonists can
be screened by contacting SGRF and a test compound with
PHF-activated premature T cells, and then selecting compounds which
suppress STAT4 phosphorylation.
[0050] Autoimmune diseases as mentioned above include, multiple
sclerosis, chronic rheumatoid arthritis, autoimmune colitis,
psoriasis, Crohn's disease, ulcerative colitis, encephalomyelitis,
polymyositis, dermatomyositis, chronic inflammatory demyelinating
polyradiculitis, insulin-dependent diabetes, spontaneous
thrombocytopenic peliosis, systemic lupus erythematosus, autoimmune
hemolytic anemia, myasthenia gravis, Kawasaki disease, and habitual
abortion. Inflammatory diseases include, for example, asthma,
allergies, hepatitis, arthritis (spinal arthritis and chronic
rheumatoid arthritis), gouty arthritis, asteoarthritis, bronchitis,
menstrual cramps, tendonitis, bursitis, dermatitis, rash,
psoriasis, burn, inflammatory bowel disease, Crohn's disease,
gastritis, irritable colon syndrome, ulcerative colitis,
polyarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's
disease, scleroderma, rheumatoid fever, sarcoidosis, nephrosis
syndrome, Behcet's syndrome, polymyositis, gingivitis,
hypersensitivity, conjunctivitis, and myocardial ischemia.
[0051] "SGRF" in the therapeutic agents of the present invention
can be prepared as a natural protein, or as a recombinant protein
obtained by well-known genetic recombination techniques. There is
no restriction as to the organism from which the "SGRF" of the
therapeutic agents of the present invention can be derived. When
SGRF is used for the treatment and prevention of human disease, it
is preferably derived from mammals, and most preferably from
humans. SGRF can be prepared as a natural protein by using
anti-SGRF antibody-coupled affinity chromatography, using extracts
from tissues expected to express SGRF, such as, testis, lymph
nodes, and the thymus.
[0052] A recombinant protein can be prepared, for example, as a
recombinant polypeptide by methods well known to those skilled in
the art. The recombinant polypeptide can be prepared by inserting a
DNA (for example, a DNA encoding the SGRF gene) into an appropriate
expression vector, transfecting this vector into appropriate host
cells, collecting transformants thus obtained, and then purifying
the polypeptide after obtaining an extract using chromatography
such as ion exchange chromatography, reverse phase chromatography,
gel filtration chromatography, or affinity chromatography, wherein
antibodies against the mutant of this invention are fixed onto a
column. A number of these columns may be used in combination.
[0053] When SGRF is expressed in a host cell (for example, an
animal cell, E. coli, etc.) as a polypeptide fused with glutathione
S-transferase protein, or as a recombinant polypeptide combined
with a number of histidines, the expressed recombinant polypeptide
can be purified using a glutathione column or a nickel column.
[0054] When E. coli is the host cell, there is no limitation as to
the above-described vector, as long as it comprises an "ori" and a
marker gene, where the "ori" is for amplifying and mass-producing
the vector in E. coli (e.g., JM109, DH5.alpha., HB101, or XL1Blue),
and the marker gene is for selecting the transformed E. coli (e.g.,
a drug-resistance gene can be selected using a drug (e.g.,
ampicillin, tetracycline, kanamycin, or chloramphenicol)). For
example, M13-series vectors, pUC-series vectors, pBR322,
pBluescript, pCR-Script, and such can be used. In addition to these
vectors, pGEM-T, pDIRECT, pT7, and the like can also be used for
subcloning and excising the cDNA. When using a vector to produce
SGRF, an expression vector is especially useful. When the
expression vector is expressed in E. coli, it should comprise the
above characteristics in order to be amplified in E. coli.
Additionally, when E. coli such as JM109, DH5.alpha., HB101 or
XL1-Blue are used as the host cell, the vector should comprise a
promoter which can efficiently promote expression of the desired
gene in E. coli, e.g., the lacZ promoter (Ward et al. (1989) Nature
341:544-546; (1992) FASEB J. 6:2422-2427), araB promoter (Better et
al. (1988) Science 240:1041-1043), or T7 promoter. Other examples
of the vectors include pGEX-5X-1 (Pharmacia), "QIAexpress system"
(QIAGEN), pEGFP, and pET.
[0055] The vector may comprise a signal sequence for secreting the
polypeptide. The signal sequence for secretion of the polypeptide
into the periplasm of E. coli can be the pelB signal sequence (Lei,
S. P. et al. (1987) J. Bacteriol. 169:4379). For example,
electroporation or the calcium, chloride method may be used to
introduce the vector into host cells.
[0056] Other vectors for use in producing SGRF, other than those
derived from E. coli, include expression vectors derived from
mammals (e.g., pCDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids Res.
(1990) 18(17):5322), pEF, pCDM8), insect cells (e.g., "Bac-to-Bac
baculovirus expression system" (GIBCO-BRL), pBacPAK8), plants
(e.g., pMH1, pMH2), animal viruses (e.g., pHSV, pMV, pAdexLcw),
retroviruses (e.g., pZIPneo), yeasts (e.g., "Pichia Expression Kit"
(Invitrogen), pNV11, SP-Q01), and Bacillus subtilis (e.g., pPL608,
pKTH50).
[0057] In order to express proteins in animal cells such as CHO,
COS, and NIH3T3 cells, the vector must comprise a promoter
necessary for expression in such cells (e.g., the SV40 promoter
(Mulligan et al. (1979) Nature 277:108), MMLV-LTR promoter,
EF1.alpha. promoter (Mizushima et al. (1990) Nucleic Acids Res.
18:5322), CMV promoter, etc.). The vector preferably comprises an
additional marker gene for selecting transformants (for example, a
drug resistance gene can be selected using a drug (e.g., neomycin,
G418, etc.)). Examples of vectors with such characteristics include
pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13, etc.
[0058] Systems for in vivo polypeptide production include those
using animals and plants. A DNA encoding SGRF can be introduced
into this animal or plant, where the SGRF is produced in vivo and
then recovered.
[0059] Animals for use in the production system described above
include mammals and insects. Mammals such as goats, pigs, sheep,
mice and cattle may be used (Vicki Glaser (1993) SPECTRUM
Biotechnology Applications). Alternatively, the mammals may be
transgenic animals.
[0060] For instance, a DNA encoding SGRF may be prepared as a
fusion gene with a gene such as goat .beta. casein gene that
encodes a polypeptide specifically produced in milk. DNA fragments
comprising the fusion gene are injected into goat embryos, which
are then introduced back into female goats. SGRF can be obtained
from milk produced by the transgenic goats born from those that
received the modified embryos, or from their offspring. Appropriate
hormones may be administered to increase the amount of
polypeptide-containing milk produced by the transgenic goats
(Ebert, K. M. et al., (1994) Bio/Technology 12:699-702).
[0061] Alternatively, insects such as the silkworm may be used.
Baculoviruses, into which a DNA encoding SGRF has been inserted,
can be used to infect silkworms, and SGRF can then be recovered
from the body fluid and silk (Susumu M. et al., (1985) Nature
315:592-594).
[0062] When using plants, tobacco can be used as an example. In the
case of tobacco, a DNA encoding SGRF may be inserted into a plant
expression vector such as pMON 530, which is introduced into
bacteria such as Agrobacterium tumefaciens. These bacteria are then
used to infect tobacco plants such as Nicotiana tabacum, and
desired mutants are recovered from the leaves (Julian K.-C. Ma et
al., Eur. J. Immunol. 24:131-138 (1994)).
[0063] SGRF obtained as above may be isolated inside or outside
(e.g., from the medium) of host cells, and purified as a
substantially pure homogeneous polypeptide. The method for
polypeptide isolation and purification is not limited to any
specific method. In fact, any standard method may be used. For
instance, column chromatography, filters, ultrafiltration, salting
out, solvent precipitation, solvent extraction, distillation,
immunoprecipitation, SDS-polyacrylamide gel electrophoresis,
isoelectric point electrophoresis, dialysis, and recrystallization
may be appropriately selected and combined to isolate and purify
the polypeptide.
[0064] SGRF of the present invention may be optionally modified or
partially deleted by treatment with an appropriate
protein-modifying enzyme, before or after purification. For
example, trypsin, chymotrypsin, lysylendopeptidase, protein kinase,
glucosidase, and the like can be used as protein-modifying
enzymes.
[0065] The above-mentioned anti-SGRF antibody of the present
invention is not restricted, and monoclonal or polyclonal
antibodies may be used. In addition, antiserum obtained by
immunizing an animal such as a rabbit with SGRF, all classes of
polyclonal and monoclonal antibodies, human antibodies, and
humanized antibodies produced by genetic recombination, are all
included in the above anti-SGRF antibody. The above-mentioned
antibodies can be prepared by the following methods. For example, a
polyclonal antibody can be prepared as follows: Small. animals such
as rabbits are immunized with SGRF to obtain serum, a serum
fraction which recognizes only SGRF is collected using SGRF-coupled
affinity column chromatography, and immunoglobulin G or M are then
purified from this fraction using protein A or protein G columns to
obtain polyclonal antibodies. A monoclonal antibody can be prepared
as follows: Small animals such as mice are immunized with SGRF, the
spleen is excised from the animal and gently crushed to separate
cells, these cells are fused with mouse myeloma cells using
reagents such as polyethylene glycol, and clones that produce
antibodies against SGRF are selected from these fused cells
(hybridomas). Next, the obtained hybridomas are transplanted into
the peritoneal cavity of a nude mouse, and ascites are collected
from the mouse. Monoclonal antibodies can be prepared by purifying
these ascites using, for example, ammonium sulfate precipitation,
protein A or protein G column chromatography, DEAE ion exchange
chromatography, or SGRF-coupled affinity column chromatography. The
above-mentioned antibodies can be used for purification and
identification of SGRF, and can be used as drugs to control SGRF
function. Human antibodies or humanized antibodies are effective
when using antibodies as therapeutic drugs for humans, taking
antigenicity into account. Human antibodies or humanized antibodies
can be prepared by methods well known to those skilled in the art.
For example, human antibodies can be prepared by using SGRF to
immunize mice whose immune system has been replaced with a human
immune system. Alternatively, human antibodies can be prepared by
panning from a phage library comprising human antibodies. Humanized
antibodies can be obtained, for example, using CDR graft methods in
which the antibody gene is cloned from monoclonal
antibody-producing cells, and its antigenic determinant site is
grafted into an existing human antibody.
[0066] The present invention also provides a method of screening
for candidate therapeutic drug compounds to treat autoimmune or
inflammatory disease. The screening method of the present invention
includes the following steps: (a) contacting SGRF with a test
compound; (b) measuring SGRF binding activity with a test compound;
and (c) selecting compounds that bind to SGRF.
[0067] The screening method of the present invention can also be
used for screening therapeutic drugs for encephalopathy.
[0068] Test compounds that can be used for screening are not
restricted. For example, synthetic small molecule compound
libraries, purified proteins, gene library expression products,
synthetic peptide libraries, cell extracts, and cell culture
supernatants can be used. SGRF binding activity with a test
compound can be measured using methods well known to those skilled
in the art.
[0069] Screening for proteins that bind to SGRF may be carried out
using, for example, West-Western blotting (Skolnik E Y, Margolis B,
Mohammadi M, Lowenstein E, Fischer R, Drepps A, Ullrich A, and
Schlessinger J (1991) Cloning of P13 Kinase-associated p85
utilizing a novel method for expression/cloning of target proteins
for receptor tyrosine kinases. Cell 65, 83-90). Specifically, phage
vectors (.lambda.gt11, ZAP11, etc) are used to construct a cDNA
library from cells or tissues (for example, the testis, lymph node,
or thymus) in which a protein binding to a protein of the present
invention is expected to be expressed. This library is then
expressed on LB-agarose and transferred to a filter membrane, which
is then reacted with purified biotin-labeled labeled or GST-fused
protein of the present invention. The plaques expressing proteins
that bind to a protein of the present invention can be detected by
using streptavidin, anti-GST antibody, or the like.
[0070] Alternatively, screening for SGRF-binding proteins or genes
encoding these proteins can be performed according to "a two-hybrid
system" ("MATCHMAKER Two-Hybrid System", "Mammalian MATCHMAKER
Two-Hybrid Assay Kit", "MATCHMAKER One-Hybrid System" (all products
by Clontech), "HybriZAP Two-Hybrid Vector System" (Stratagene),
literature [Dalton S., and Treisman R., Characterization of SAP-1,
a protein recruited by serum response factor to the c-fos serum
response element. Cell 68, 597-612 (1992)]). The two-hybrid system
can be performed as follows: SGRF is fused to an SRF- or
Gal4-binding domain, and expressed in yeast cells; a cDNA library,
which expresses proteins as fusion proteins with the VP16 or Gal4
transcription activation domain, is prepared from cells expected to
express proteins binding to SGRF; the library is introduced to the
above-mentioned yeast cells; and library-derived cDNA are isolated
from the positive clones detected (positive clones can be confirmed
by reporter gene activation due to the binding of SGRF and the
protein when expressed in the yeast cells). The protein encoded by
the isolated cDNA can be obtained by introducing and expressing
that cDNA in E. coli.
[0071] Screening for SGRF-binding proteins can also be performed by
applying cell extracts or culture supernatants expected to express
SGRF-binding proteins, onto a SGRF-coupled affinity chromatography
column, and purifying proteins that specifically bind to this
column.
[0072] Small molecule compounds, proteins (or genes encoding these
proteins), peptides, and such, which bind to SGRF can be isolated
using methods well known to those skilled in the art. Such methods
include, for example, screening for binding-molecules by contact
with synthetic compounds, natural product banks, or random phage
peptide display libraries with an immobilized SGRF; and
high-throughput screening methods using combinatorial chemistry
techniques (Wrighton N C; Farrell F X; Chang R; Kashyap A K;
Barbone F P; Mulcahy L S; Johnson D L; Barrett R W; Jolliffe L K;
Dower W J., Small peptides as potent mimetics of the protein
hormone erythropoietin, Science (United States) July 26, 273
p458-64 (1996) Verdine G L., The combinatorial chemistry of nature.
Nature (England) November 7, 384 p11-13 (1996), Hogan J C Jr.,
Directed combinatorial chemistry. Nature (England) November 7, 384
p17-9 (1996)).
[0073] The present invention also provides methods of screening for
compounds that can substitute for SGRF protein function, where the
methods use genetically modified animals of the present invention.
The above-mentioned screening methods include the following steps:
(a) administrating a test compound to a genetically modified animal
of the present invention; (b) determining whether the test compound
substitutes for SGRF function; and (c) selecting compounds that can
substitute for SGRF function.
[0074] Administration of the test compound to a genetically
modified animal of (a) can be performed orally or parenterally.
Moreover, in determining whether a test compound is substitutes for
SGRF function, for example, the following conditions are determined
to substitute for SGRF function:
[0075] (1) on administration of the test compound, a genetically
modified animal of the present invention originally susceptible to
pathogenic microbes such as Listeria, gains resistance to such
microbes;
[0076] (2) on administration of the test compound, symptoms of
autoimmune diseases such as EAE are induced in a genetically
modified animal of the present invention, although induction of
such autoimmune diseases in the animal was originally
difficult;
[0077] (3) on administration of the test compound, a genetically
modified animal of the present invention originally displaying mild
symptoms of inflammatory disease, such as delayed-hyper
sensitivity, develops an inflammatory disease or the symptoms are
worsened;
[0078] (4) jak or STAT is phosphorylated when a test compound is
added to cultured cells derived from primary or passaged cultures
of SGRF receptor-expressing cells (for example, human T cell
blastocysts), taken from a genetically modified animal of the
present invention (or a normal animal);
[0079] (5) IL-23 dependent proliferation of IL-12R.beta.1- and
IL-23R-transfected Ba/F3 cells is specifically inhibited. (When
IL-12R.beta.1 and IL-23R are transfected into Ba/F3 cells, these
cells proliferate by reacting to IL-23.)
[0080] Whether resistance to pathogen infection has been conferred
in the above-mentioned (1) can be assessed, for example, by
comparing pathogen infection of mice in which both SGRF gene
alleles are inactivated, with that of normal individuals in which
SGRF is not inactivated (SGRF/IL-23.sup.+/+). Listeria and such are
often used for this type of mouse infection study. However,
infections by pathogenic microbes other than Listeria, such as
pathogenic E. coli, Salmonella, and Staphylococcus, or viruses such
as Vascular Stomatitis Virus (VSV), Lymphocytic Choriomeningitis
Virus (LCMV), and influenza, or parasites such as Leishmania and
Japanese Schistosomiasis, can be used for comparison. The
above-described pathogenic microbes used for assessment of SGRF
involvement in defense mechanisms are not limited to those that
infect mice. Microbes that can infect other animals, such as
HTLV-I, HCV, HBV or HIV, can also be used for assessment.
[0081] A case such as that above in (2) can be assessed by using
well-known immunological methods to induce a disease model in a
genetically modified mice of the present invention, and then
comparing these with normal individuals. For example, experimental
autoimmune encephalomyelitis (EAE) is known to be a model for human
multiple sclerosis. Collagen-induced arthritis is known as a model
for human chronic rheumatoid arthritis, and Dextran sodium
sulfate-induced colitis is known as a model for colitis. Assessment
can also be performed using a human systemic erythematodis model or
a diabetes model, by breeding with known lpr/lpr mice, which
develop autoimmune disease naturally, or with NOD mice or the
like.
[0082] In the above-mentioned (3), assessment can be performed by
using well-known immunological methods to induce a disease model in
a genetically modified mice of the present invention, and then
comparing it with a normal mouse. For example, an asthma model can
be established by re-sensitizing OVA through the respiratory tract
after immunization. Alternatively, hepatitis can be induced by
injecting concanavallin A into mouse-tail veins. Delayed Type
Hypersensitivity (DTH) is a general inflammation model.
[0083] SGRF, antagonists against SGRF, anti-SGRF antibodies, or
compounds that can be isolated using the screening methods of the
present invention, can be used as pharmaceutical agents for humans
and non-human animals by directly administering the isolated
compound to the patient, or by formulating and administering it
according to pharmacologically accepted methods. For example, the
compound(s) may be orally administered as tablets (sugarcoated as
necessary), capsules, elixirs, or microcapsules. It may also be
parenterally administered as injections of an aseptic solution or
suspension with water or another pharmaceutically acceptable
liquid. For example, by suitably mixing a pharmaceutical
composition with a pharmacologically acceptable carrier or medium,
a unit dosage form such as one generally required for drug
implementation can be formulated. Specific examples of a
pharmacologically acceptable carrier or medium include sterilized
water, physiological saline, vegetable oil, an emulsifier,
suspension agent, surfactant, stabilizer, flavoring agent,
excipient, vehicle, antiseptic, binder, and so on. The amount of
active ingredient in these preparations is adjusted to obtain a
suitable volume within a specified range.
[0084] Examples of additives that can be mixed into tablets and
capsules include binders such as gelatin, cornstarch, gum
tragacanth, and gum arabic; excipients such as crystalline
cellulose; swelling agents such as cornstarch, gelatin, and alginic
acid; lubricants such as magnesium stearate; sweeteners such as
sucrose, lactose, and saccharin; and flavoring agents such as
peppermint, Gaultheria adenothrix oil, and cherry flavoring. When
the preparation unit is in the form of a capsule, liquid carriers
such as fats and oils can be included in addition to the above
materials. Aseptic compositions for injections can be formulated
according to ordinary preparation methods, using vehicles such as
distilled water for injection.
[0085] Examples of aqueous solutions suitable for injections
include physiological saline, and isotonic liquids containing
glucose or other adjuvants, such as D-sorbitol, D-mannose,
D-mannitol, and sodium chloride. These may be used in combination
with suitable solubilizers, for example, alcohols such as ethanol;
polyalcohols including propylene glycol and polyethylene glycol;
and nonionic surfactants such as Polysorbate 80 (TM) and
HCO-50.
[0086] Examples of oleaginous liquids include sesame oil and
soybean oil, and these may be used in combination with
-solubilizers such as benzyl benzoate and benzyl alcohol. In
addition, the blend may include buffers such as phosphate buffer
and sodium acetate buffer; analgesics such as procaine
hydrochloride; stabilizers such as benzyl alcohol and phenol; and
antioxidants. The prepared injection liquid is normally filled into
suitable ampules.
[0087] Administration to patients can be carried out using standard
methods known to those skilled in the art, such as, by
intra-arterial injection, intravenous injection or subcutaneous
injection; or by intranasal, transbronchial, intramuscular,
percutaneous or oral administration. Dosage varies depending on a
patient's body weight and age, administration method, and so on;
however, an appropriate dosage can be suitably selected by a person
skilled in the art. If the compound can be encoded by a DNA, gene
therapy may also be carried out by incorporating the DNA into a
vector for gene therapy. Dosage and administration method vary
according to a patient's body weight, age, and symptoms, but can be
suitably selected by a person skilled in the art.
[0088] Although compound dosage varies according to symptoms,
orally administered dosage for an adult (with a body weight of 60
kg) typically ranges from about 0.1 to 100 mg per day, preferably
about 1.0 to 50 mg per day, and more preferably about 1.0 to 20 mg
per day.
[0089] Although parenterally administered dosage varies according
to the target organ, symptoms, administration method, and subject
to be administered, a single dosage in the form of an injection
preparation for an adult of body weight 60 kg, for example, is
normally about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg
per day, and more preferably about 0.1 to 10 mg per day.
Administration is preferably carried out by intravenous injection.
When administering to other animals, doses adjusted per 60 kg body
weight or per body surface area can be administered.
BRIEF DESCRIPTION OF THE DRAWINGS
[0090] Wild type mice in the figures are represented as Wt.
SGRF/IL-23.sup.-/- mice are represented as KO.
[0091] FIG. 1 shows the genomic nucleotide sequence encoding the
mouse SGRF gene.
[0092] FIG. 2 shows the continuation of the genomic nucleotide
sequence encoding the mouse SGRF gene in FIG. 1.
[0093] FIG. 3 depicts a photograph and a drawing, showing SGRF mRNA
expression analysis in mouse tissue and the construction of
SGRF/IL-23.sup.-/- mice.
[0094] a. A photograph depicting the results of Northern blot
analysis of mouse SGRF transcripts. PolyA.sup.+ RNA (5 .mu.g) used
for Northern blot hybridization was prepared from tissues (brain,
heart, thymus, lung, liver, spleen, lymph nodes, kidney, and
testis) of C57BL/6 mouse (male, four weeks old). .beta.-Actin was
used as a reference.
[0095] b. A drawing depicting the structures of the mouse SGRF gene
and gene-targeting vector. A targeting vector was generated by
replacing two SGRF gene exons with a neomycin resistant gene (neo),
located between loxP sites. The diphtheria toxin A (DT-A) fragment
gene is placed outside of the 3' genomic DNA fragment. In the
figure, exons are filled boxes, and loxP sites are filled
triangles.
[0096] c. A photograph depicting the result of southern blot
analysis of EcoR I-digested genomic DNA. After homologous
recombination, the mutated allele was detected using an EcoRI
cleavage site introduced into the neo gene.
[0097] d. A photograph showing the results of RT-PCR analysis of
polyA.sup.+ RNA isolated from thymus. The photograph shows PCR
products of SGRF from mice of each SGRF genotype and those of GAPDH
as a control.
[0098] FIG. 4 shows graphs of antigen-induced cytokine production
and proliferation by lymph nodes cells collected from
SGRF/IL-23.sup.-/- and wild type mice. Groups of five mice (male,
ten weeks old), were each immunized with KLH (200 .mu.g) in CFA.
Lymph nodes were harvested five days later, cultured for 48 hours
in 300 .mu.g/ml KLH, and then analyzed for their ability to produce
IFN-.gamma. (a), IL-4 (b), IL-5 (c), or IL-10 (d), or to
proliferate (e). ConcanavalinA (ConA, 2 .mu.g/ml) was used as the
control for non-specific proliferation. Data are presented as the
mean .+-.SD values derived from the five mice in each group.
[0099] .dagger., P<0.003; .dagger-dbl., p<0.05; and .sctn.,
p<0.03 (comparison between SGRF/IL-23.sup.-/- and wild type mice
by unpaired t-test).
[0100] FIG. 5 depicts graphs representing susceptibility to L.
monocytogenes infection, and DTH reactions, in wild type and
SGRF/IL-23.sup.-/- mice.
[0101] a. Survival of SGRF/IL-23.sup.-/- or wild type mice after
infection with L. monocytogenes. Groups of six mice (female, ten
weeks old) were intravenously injected with 2.3.times.10.sup.6 CFU
of L. monocytogenes.
[0102] b. Multiplication of L. monocytogenes in SGRF/IL-23.sup.-/-
or wild type mice. Four days after infection (3.1.times.10.sup.6
CFU), livers were collected and bacteria number determined. The
Mann-Whitney rank-test was used for statistical analysis
(P<0.004).
[0103] c. Delayed-type hypersensitivity (DTH) reaction in
SGRF/IL-23.sup.-/- and wild type mice. Six mice (male, ten weeks
old) were subcutaneously immunized with 50 .mu.l of CFA solution
comprising mBSA (2.5 mg/ml). After five days, one rear footpad of
each mouse was injected with 30 .mu.l of 5 mg/ml mBSA.
SGRF/IL-23.sup.-/- and wild type mice were compared at each time
point using unpaired t-tests (P<0.007).
[0104] FIG. 6 depicts a graph and a photograph representing the
result of EAE induction in wild type and SGRF/IL-23.sup.-/- mice.
Mice (female, eight weeks old) were immunized with MOG peptide
35-55 in CFA, and subcutaneously injected with pertussis toxin
according to the protocol for EAE induction.
[0105] a. A graph showing the clinical course of average disease
scores in wild type mice (n=6) and SGRF/IL-23.sup.-/- mice (n=6).
The scores of four of six wild mice that died are included in the
graph throughout the experiment. The remaining mice survived.
[0106] b to e. Photographs representing histological sections of
CNS from wild type and SGRF/IL-23.sup.-/- mice in which EAE was
experimentally induced (b,d: wild type mice; c,e:
SGRF/IL-23.sup.-/- mice). Sections were recovered one month after
the mice were sensitized. Tissue sections (4 to 6 .mu.m thick) were
stained with hematoxylin and eosin (.times.200) (FIG. 6b and 6c) or
with Kluver-Barrera (.times.200) (FIG. 6d and 6e). The arrows show
infiltration of lymphocytes and the arrowhead shows
demyelination.
BEST MODE FOR CARRYING OUT THE INVENTION
[0107] The present invention will be described below in detail with
reference to Examples, but it is not to be construed as being
limited thereto.
EXAMPLE 1
Expression Analysis of SGRF mRNA
[0108] Expression analysis of SGRF mRNA in mouse tissue was
performed by Northern blotting using SGRF cDNA as a probe. High
expression of mouse SGRF mRNA was confirmed in the thymus, and
expression was detected in the brain and testis (FIG. 3a). RT-PCR
analysis also detected mRNA expression in the spleen. and lymph
nodes (data not shown).
EXAMPLE 2
Construction of the Mice
[0109] Cloning of the SGRF gene was performed according to the
method described in Patent EP1072610. The mouse SGRF genome was
screened by Genome Systems, Inc. (now Incyte Genomics, Ins. St.
Louis, USA). Two BAC (Bacteria Artificial Chromosome) clones were
obtained. The Clone Addresses and GS Control Numbers identified by
Genome System, Inc. are as follows:
1 Clone Address GS Control Number 225 (L12) 24057 198 (A03)
24058
[0110] A large number of BAC clones were produced by known methods,
and BAC DNAs were prepared using a Large-Construction Kit
(QIAGEN).
[0111] Restriction digest mapping suggested that these BAC clones
were identical to each other. However, a .about.13 Kbp Hind III
fragment was produced by digesting the GS Control Number 24057
clone, and a .about.9 Kbp Bgl II fragment was produced by digesting
the GS Control Number 24058 clone. The .about.13 Kbp fragment was
subcloned into pBluescript vector (STRATAGENE) at the Hind III
site, and the .about.9 Kbp fragment was cloned into a pGEM-T-easy
vector (Promega) at the EcoRV site (converted to Bgl II). Vectors
carrying each DNA fragment were referred to as pBSK-mSGRF (Hind
III) and pGEM-mSGRF (Bgl II) respectively. The genomic sequence
encoding the mouse SGRF (mSGRF) gene was determined using sequence
analysis and is shown in FIG. 1 and 2, and in SEQ ID NOs: 1 to 7
(note this sequence is that of 129/SV JII mice).
[0112] The above-mentioned sequence data results revealed that both
the mouse SGRF (mSGRF) gene and the human SGRF (hSGRF) gene
(GenBank Accession No. AB030001) comprise four exons. Therefore, a
genetically modified mouse of the present invention can be
constructed as follows: A targeting vector is constructed by
replacing the SGRF exon region with an appropriate drug marker
gene. The targeting vector is then transfected into mouse ES cell
strains using electroporation or the like, and cell strains that
underqo homologous recombination are selected.
[0113] Specifically, this was performed as follows:
[0114] The mouse SGRF DNA fragment inserted into pGEM-mSGRF (Bgl
II) contained a .about.9 Kbp promoter region, which included a
region from the Bgl II site 18 bases upstream of the start codon to
the Bgl II site located further upstream. pBSK-mSGRF (Hind III)
also contained a DNA fragment that included a region from
.about.1.7 Kbp of the start codon to 13 Kbp downstream. The
.about.1.1 Kbp region between Intron 2 and Exon 4 (sequence data:
from 5'-CTAAGCCGATGTTGATGTGTC-3' (SEQ ID NO: 8) to
5'-GCAGATGCACAGTACTCCAGACAGC-3' (SEQ ID NO:9)) was amplified by PCR
using pBSK-mSGRF (Hind II) as a template, and the primers described
below (FmSGREX2 and RSGR-A), which were designed to create
restriction sites (Sal I, Pst I, and Xba I). The PCR product was
then TA-cloned into a pGEM-T-Easy vector, and the vector was
digested using Sal I and Xho I.
[0115] Forward primer:
[0116] FmSGREX2: 5'-GTC GAC TGC AGT CTA GAC TAA GCC GAT GTT GAT GTG
TC-3' (SEQ ID NO: 10)
[0117] Reverse primer:
[0118] RSGR-A: 5'-GCT GTC TGG AGT ACT GTG CAT CTG C-3' (SEQ ID NO
11)
[0119] In this case, a knockout vector was constructed using a
.about.0.9 Kbp fragment (produced by digestion at Sal I and Xho I
sites in the FmSGRFEX2 primer and in Exon 4 of the SGRF gene), and
the above-described .about.9 Kbp fragment of the mSGRF promoter
region inserted into pGEM-mSGRF (Bgl II). Cell strains which did
not undergo homologous recombination were eliminated by inserting a
drug resistance gene (neo) between these fragments, and by adding a
diphtheria toxin A fragment (DT-A).
[0120] In this Example, as described above, the SGRF gene was
inactivated by replacing Exon 1 and 2 of the SGRF gene with a drug
resistance gene (neo) (FIG. 3(A)). Since this drug resistance gene
(neo) would probably influence the expression of neighboring genes,
a loxP sequence (5'-ATA ACT TCG TAT AGC ATA CAT TAT ACG AAG TTA
T-3' (SEQ ID NO: 12)) was inserted at either side of the neo
sequence. Thus, the neo can be deleted using a Cre protein, if a
known or unknown gene be located near the SGRF gene. The targeting
vector was digested at a single site using a restriction enzyme
(Not I), and then transfected into ES cells by electroporation.
[0121] Homologous recombinants were constructed by transfecting
AB2.2 cells (ES cells derived from 129SvEv mice) according to the
manual attached to The Mouse Kit (Lexicon Genetics Inc.),
commercially available from TAKARA. Homologous recombinants were
selected by G418. PCR was used to screen for recombinants. ES cells
used for screening were cultured in a 96-well plate. Each well was
washed twice with 200 .mu.l PBS and treated with cell lysis buffer
(5 .mu.l 10.times.LA buffer (use for TAKARA LA Taq), 5 .mu.l 5%
NP-40, 4 .mu.l proteinase K (TAKARA, 20 mg/l), 35 .mu.l distilled
water) for two hours at 55 degrees Celsius. These cells were
incubated at 95 degrees C. for 15 minutes to inactivate proteinase
K, and then used for PCR samples. The PCR reaction mixture
contained 1 .mu.l of an above-mentioned PCR sample, 5 .mu.l
10.times.LA buffer, 5 .mu.l MgCl.sub.2 (25 mM), 8 .mu.l dNTP (2.5
mM), 0.4 .mu.l each primers (50 .mu.M each), 0.5 .mu.l LA taq
(TAKARA), and 29.7 .mu.l distilled water (total 50 .mu.l). PCR was
performed under the following condition: preheating at 95 degrees
C. for one minute; 35 cycles of amplification at 95 degrees C. for
30 seconds, 64 degrees C. for 30 seconds, and 72 degrees C. for 80
seconds; and heat extension at 72 degrees C. for seven minutes.
[0122] The primers were as below. A .about.1.3 Kbp band was
amplified in ES cell samples in which homologous recombination
occurred. Primer Fneo-1 was in the drug resistance gene in the
knockout vector, and primer RSFR-A was in Exon 4 of the SGRF gene,
which was located outside of the knockout vector.
[0123] Reverse primer:
[0124] RSGR-A: 5'-GCT GTC TGG AGT ACT GTG CAT CTG C-3' (SEQ ID NO:
13)
[0125] Forward primer:
[0126] FNeo-1: 5'-CCG TGA TAT TGC TGA AGA GCT TGG C-3' (SEQ ID NO:
14)
[0127] 420 and 760 ES cell clones were analyzed in the two
experiments. Of these, two and six clones respectively were
homologous recombinants. Thus the recombination efficiencies were
2/420 (0.467%) and 6/760 (0.79%), and the combined total
recombination efficiency for both experiments was 0.68%.
[0128] Chimeric mice were obtained by injecting seven ES cell
strains, in which one SGRF gene allele was inactivated, into
blastocysts derived from C57BL/6J mice. Mice comprising cells in
which one SGRF gene allele was inactivated (hereafter referred to
as "SGRF/IL-23.sup.+/- mice") were obtained by mating C57BL/6 mice
with the above-described chimeric mice. SGRF/IL-23.sup.+/- mice
were obtained in four strains, clone numbers 11, 23, 75, and 86, of
the seven cell strains used for the experiment. Mice derived from
clone 11 and clone 86 were used for further studies. Mice in which
both SGRF gene alleles were inactivated (hereafter referred to as
"SGRF/IL-23.sup.-/- mice") were obtained by mating the
SGRF/IL-23.sup.+/- mice with each other. Southern blot analysis was
used to confirm the insertion of the mutant gene into one or both
SGRF gene alleles in mice thus obtained (FIG. 3(c)).
[0129] Loss of gene expression was confirmed by a reverse
transcriptase polymerase chain reaction (RT-PCR) using
thymus-derived polyA.sup.+ RNA (FIG. 3(d)). Both heterologous and
homologous SGRF/IL-23 mice showed normal growth and appearance, and
were able to reproduce.
EXAMPLE 3
Analysis of SGRF Gene-deleted Mice
[0130] (1) Cytokine Production in SGRF/IL-23.sup.-/- Mice Immunized
with KLH
[0131] The ability of lymph node cells, obtained from keyhole
limpet haemocyanin (KLH)-immunized wild type mice (normal mice,
SGRF/IL-23.sup.+/+) and SGRF/IL-23.sup.-/- mice, to produce
cytokines and to proliferate was examined in order to investigate
the involvement of SGRF/IL-23 in antigen-induced cytokine
production and ability to proliferate T cells.
[0132] To induce a Th1 response, mice were immunized subcutaneously
at the base of their tail using 200 .mu.g KLH in PBS, in a 1:1
emulsion with complete Freund's adjuvant (CFA), containing 5 mg/ml
Mycobacterium tuberculosis strain H37Ra, (Difco Laboratories).
After five days, inguinal lymph nodes were removed and the cells
were cultured in RPMI1640 supplemented with 10% FCS and 0.3 mg/ml
of KLH. To measure cytokine production, lymph node cells were
cultured in 1 ml of medium in 24-well plates until the cells
proliferated to 6.times.10.sup.6 cells/ml. After culturing for 48
hours, culture supernatants were harvested and cytokine levels were
determined using enzyme-linked immunosorbent assay (ELISA) kits,
obtained from R&D Systems, according to the manufacturer's
instructions. Furthermore, radiolabel incorporation into DNA was
measured by MicroBeta scintillation counting (Perkin Elmer) as
follows:
[0133] After 48 hours of incubation, 0.25 .mu.Ci of
[.sup.3H]thymidine (Amersham Pharmacia) was added and incubated for
four hours in 96-well flat-bottomed microplates in which there were
6.times.10.sup.6 cells/ml in a total volume of 200 .mu.l. Then, the
degree of proliferation was measured.
[0134] IFN-.gamma. production by SGRF/IL-23-deficient lymphocytes
was significantly impaired compared to the production by wild-type
lymphocytes (FIG. 4a), whereas KLH-induced IL-4 and IL-5 production
was markedly enhanced (FIG. 4b,c). In contrast, both IL-10
production and KLH-induced proliferation were similar for wild type
and SGRF/IL-23-deficient lymphocytes (FIG. 4d,e).
[0135] These data indicate that in SGRF/IL-23.sup.-/- mice,
production of Th1-type cytokines is reduced, and production of
Th2-type cytokines is increased.
[0136] (2) Defending the Body Against Pathogenic Microbes and
DTH
[0137] Th1-type responses are important in defenses against
intracellular pathogens such as Listeria, and Th1-type cytokines
such as IFN-.gamma. and TNF-.alpha. are essential for this defense
(Kaufmann S H. Immunity to intracellular bacteria. Annu. Rev
Immunol 11, 129-163 (1993)).
[0138] The survival rate of SGRF/IL-23.sup.-/- mice after Listeria
infection, and the number of Listeria present in the liver four
days after Listeria infection, were investigated to examine the
involvement of SGRF/IL-23 in biophylaxis against pathogenic microbe
infection.
[0139] Specifically, Listeria (serum type 4b) (2.3.times.10.sup.6
CFU/mouse) were injected intravenously (Suzuki H. et al., A role
for macrophage scavenger receptors in atherosclerosis and
susceptibility to infection. Nature 386, 292-296 (1997)) into
either SGRF genotypic mice (male, ten weeks old) or wild type mice
(male, ten weeks old) and the post-infection survival rates of the
mice were examined everyday for 15 days. The results indicated the
survival rate of SGRF/IL-23.sup.-/- mice was lower than that of
wild type mice (FIG. 5a).
[0140] Listeria (3.1.times.10.sup.6 CFU/mouse) was also injected
into mice in order to examine post-infection Listeria
proliferation. Four days after infection, and under sterile
conditions, the mice livers were removed and homogenized in sterile
PBS. The homogenized tissues were diluted stepwise with PBS, plated
on brain-heart infusion agar (Difco), and incubated at 37 degrees
C. for 16 hours. CFUs were then counted. L. monocytogenes
proliferation in the livers of SGRF/IL-23.sup.-/- mice four days
after injection was significantly increased compared with that of
wild-type mice (FIG. 5).
[0141] These results suggest that SGRF/IL-23.sup.-/- mice, compared
with wild type mice, are susceptible (less resistant) to L.
monocytogenes. Thus, these results suggest that SGRF/IL-23, as well
as IL-12, contributes to the elimination of L. monocytogenes.
[0142] To further examine the role of SGRF/IL-23 on Th1-type immune
responses, the present inventors investigated delayed-type
hypersensitivity (DTH), which is the typical in vivo manifestation
of cell-mediated immunity.
[0143] Delayed-type hypersensitivity was induced by using a well
known method (Immunity, Vol.4, p471 (1996)). Specifically, mice
were immunized with methyl BSA (SIGMA) by intradermal injection of
a 50 .mu.l emulsion of 2.5 mg/ml mBSA in CFA (Beckton Dickinson) at
two sites on the abdomen. Five days after immunization, DTH was
induced in the mice by subcutaneous injection of 30 .mu.l of 5.0
mg/ml mBSA into one rear footpad. A comparable volume of PBS was
injected into the other rear footpad as a control. Measurements of
footpad swelling were taken using a micrometer (Ozaki Inc.) at 24,
48 and 72 hours after injection. Footpad swelling following methyl
BSA injection was measured by comparison with that caused by PBS
injection, and average values for each group were obtained
(n=6).
[0144] The. results showed that for three days after injection,
specific footpad swelling was suppressed in the SGRF/IL-23.sup.-/-
mice (FIG. 5c).
[0145] These results indicate that SGRF/IL-23 plays an important
role in Th1-type responses. Therefore, it is predicted that SGRF is
a factor necessary for the defense against infections caused by
pathogenic microbes.
[0146] (3) Involvement in Autoimmune Disease Development
[0147] The involvement of SGRF/IL-23 in autoimmune diseases such as
multiple sclerosis, chronic rheumatoid arthritis, and autoimmune
colitis can be investigated by inducing disease models in the
genetically modified mice of the present invention according to
known immunological methods, and then making comparisons with
normal mice.
[0148] Experimental autoimmune encephalomyelitis (EAE) is a central
nervous system (CNS) autoimmune disease mediated by Th1 responses,
and is known as a model for human multiple sclerosis. The following
experiment was performed to investigate the role of SGRF/IL-23 in
autoimmune disease.
[0149] EAE was induced in SGRF/IL-23.sup.-/- mice and wild type
mice by well known methods (J. Exp. Med. Vol. 186 p1233, 1997).
Specifically, 300 .mu.g of MOG peptide (MEV GWY RSP FSR VVH LYR
NGK/SEQ ID NO: 15; Sawady Technology) and 0.2 ml of CFA (including
500 .mu.g of Mycobacterium tuberculosis strain H37Ra (Difco
Laboratories)) was injected subcutaneously into one side of the
mice abdomens on day 0 and day 7. 200 ng of Pertussis toxin (List
Biological Laboratories) was injected into the mice veins on day 0,
and administrated intraperitoneally two days after the first
immunization.
[0150] After inducing EAE in the mice, pathology scoring was
conducted according to a known method, and using a scale from 1 to
5: 1, normal; 2, wobbly gait; 3, hind limb paralysis; 4, hind and
fore limb paralysis; 5, death. Each mouse was scored and average
values were collected. The results indicated that
SGRF/IL-23.sup.-/- mice showed significant resistance to
MOG-induced EAE compared to wild type mice. Six SGRF/IL-23.sup.-/-
mice and six wild type mice were used for the experiment in FIG. 6.
Although EAE developed in two of the six SGRF/IL-23.sup.-/- mice,
their pathology scores were low. On the other hand, all wild type
mice developed severe EAE, and four of the six wild type mice
died.
[0151] Histological analysis was performed on EAE-induced
SGRF/IL-23.sup.-/- mice and wild type mice. Spinal cords from
EAE-induced mice were removed and fixed with 20% formalin neutral
buffered solution (Wako) in order to conduct histological analysis.
Paraffin sections were prepared and stained using hematoxylin/eosin
and Kluver-Barrera staining in the Sapporo General Pathology Co.
Ltd.
[0152] Histological analysis of the spinal cords of wild-type mice
injected with MOG peptide showed typical lymphocyte infiltration
(FIG. 6b) and demyelination (FIG. 6d). In contrast, no histological
change was observed in the spinal cords of SGRF/IL-23.sup.-/- mice
(FIG. 6c, e).
[0153] These results indicate that SGRF/IL-23.sup.-/- is an
important component in the development of EAE, and that SGRF could
be a new target for multiple sclerosis (MS) therapy in humans.
[0154] (4) Involvement in the Bone Metabolism
[0155] The well known OVX model, in which ovaries are removed from
a female mouse, can be used to determine whether SGRF is involved
in the bone metabolism.
INDUSTRIAL APPLICATION
[0156] The present invention provides genetically modified animals
in which both SGRF gene alleles are inactivated and SGRF activity
has been eliminated. The genetically modified animals and ES cells
of the present invention can be used to predict the side effects of
SGRF inhibitors such as anti-SGRF antibodies or SGRF antagonists.
The genetically modified animals and ES cells of the present
invention can also be used to determine whether a test protein or
small molecule comprises the function of substituting for SGRF
function, or whether it can be used to screen for DNA that encodes
a protein comprising the function of substituting for SGRF
function.
[0157] Furthermore, since the genetically modified mice (SGRF KO
mouse) are susceptible to infection by the pathogenic microbe
Listeria, SGRF or alternative substances involved in SGRF signaling
are considered to be therapeutic agents against pathogenic
microbes.
[0158] Moreover, experimental autoimmune encephalomyelitis is not
easily induced in SGRF KO mice and delayed-hypersensitivity is
reduced. Therefore, antagonists that inhibit SGRF function are
considered to be strong candidates for therapeutic agents for
autoimmune or inflammatory diseases.
Sequence CWU 1
1
15 1 185 DNA Mus musculus 1 agatctgaga agcagggaac aagatgctgg
attgcagagc agtaataatg ctatggctgt 60 tgccctgggt cactcagggc
ctggctgtgc ctaggagtag cagtcctgac tgggctcagt 120 gccagcagct
ctctcggaat ctctgcatgc tagcctggaa cgcacatgca ccagcgggac 180 atatg
185 2 219 DNA Mus musculus 2 gtaagtgtca gctcctggga ccgcgcagaa
aaccttccca gtcctccaag tgtgtaggtt 60 taatggaagc tgtggccccg
ggtggatctg gagggttgga agccatcgtg gaatgagata 120 ggacagaaga
ctggggcttc tggaagagtt gtgggccggc ggttgagcgg aatgcaaagc 180
ggtcacctcg cctcactgtt cccactccct ccattacag 219 3 219 DNA Mus
musculus 3 gtaagtgtca gctcctggga ccgcgcagaa aaccttccca gtcctccaag
tgtgtaggtt 60 taatggaagc tgtggccccg ggtggatctg gagggttgga
agccatcgtg gaatgagata 120 ggacagaaga ctggggcttc tggaagagtt
gtgggccggc ggttgagcgg aatgcaaagc 180 ggtcacctcg cctcactgtt
cccactccct ccattacag 219 4 265 DNA Mus musculus 4 gtaccactaa
gccgatgttg atgtgtctag gagagggagg tgagaggaag ctgagcgtcc 60
atggccattt agctttgtct gagatgacga ggagccatag ttggcttgaa gccagcctga
120 gctgtgggtg gtaagtttaa ggccaaagcc taaggtagtg aaatgctgtc
taaagaaaga 180 aaaaggaaaa acagaggaag gaagaaaggc aggcaggcac
taggaaagag gatctatctg 240 tcttgattgt tttcttcttt cccag 265 5 144 DNA
Mus musculus 5 ttctgcttgc aaaggatccg ccaaggtctg gctttttata
agcacctgct tgactctgac 60 atcttcaaag gggagcctgc tctactccct
gatagcccca tggagcaact tcacacctcc 120 ctactaggac tcagccaact cctc 144
6 102 DNA Mus musculus 6 caggtatgaa ctagggatct ggaagatagg
gctagccagt gtttgaaaaa gaagctcgga 60 gcttagtatc tggagtcctt
tctgactgtg tcctgtgtct tt 102 7 884 DNA Mus musculus 7 cagccagagg
atcacccccg ggagacccaa cagatgccca gcctgagttc tagtcagcag 60
tggcagcgcc cccttctccg ttccaagatc cttcgaagcc tccaggcctt tttggccata
120 gctgcccggg tctttgccca cggagcagca actctgactg agcccttagt
gccaacagct 180 taaggatgcc caggttccca tggctaccat gataagacta
atctatcagc ccagacatct 240 accagttaat taacccatta ggacttgtgc
tgttcttgtt tcgtttgttt tgcgtgaagg 300 gcaaggacac cattattaaa
gagaaaagaa acaaacccca gagcaggcag ctggctagag 360 aaaggagctg
gagaagaaga ataaagtctc gagcccttgg ccttggaagc gggcaagcag 420
ctgcgtggcc tgaggggaag ggggcggtgg catcgagaaa ctgtgagaaa acccagagca
480 tcagaaaaag tgagcccagg ctttggccat tatctgtaag aaaaacaaga
aaaggggaac 540 attatacttt cctgggtggc tcagggaaat gtgcagatgc
acagtactcc agacagcagc 600 tctgtacctg cctgctctgt ccctcagttc
taacagaatc tagtcactaa gaactaacag 660 gactaccaat acgaactgac
aaatactacc actatgacct gtgacaaagc tgtttattta 720 ttaagtggga
agggaacttt tgatattatt tatccttgta acagtataga tgatggttat 780
ttattctatt tataaggaat tatgtatttt tttttcaata aagatttatt tatgtggctc
840 tctgggtcta aatttctaag tgtagtcggg agagaaaaga gatg 884 8 21 DNA
Mus musculus 8 ctaagccgat gttgatgtgt c 21 9 25 DNA Mus musculus 9
gcagatgcac agtactccag acagc 25 10 38 DNA Artificial Sequence
Description of Artificial SequenceArtificially Synthesized Primer
Sequence 10 gtcgactgca gtctagacta agccgatgtt gatgtgtc 38 11 25 DNA
Artificial Sequence Description of Artificial SequenceArtificially
Synthesized Primer Sequence 11 gctgtctgga gtactgtgca tctgc 25 12 34
DNA Artificial Sequence Description of Artificial
SequenceArtificially Synthesized Sequence 12 ataacttcgt atagcataca
ttatacgaag ttat 34 13 25 DNA Artificial Sequence Description of
Artificial SequenceArtificially Synthesized Primer Sequence 13
gctgtctgga gtactgtgca tctgc 25 14 25 DNA Artificial Sequence
Description of Artificial SequenceArtificially Synthesized Primer
Sequence 14 ccgtgatatt gctgaagagc ttggc 25 15 21 PRT Artificial
Sequence Description of Artificial SequenceArtificially Synthesized
Peptide 15 Met Glu Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val Val
His Leu 1 5 10 15 Tyr Arg Asn Gly Lys 20
* * * * *