JP2003180375A - Gene expression failure animal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an LLPL gene expression failure non-human animal, and to provide an LLPL gene transfer non-human animal. <P>SOLUTION: The LLPL gene-inactivated non-human animal ES cell is highly useful on the creation of an LLPL gene expression failure non-human animal. The LLPL gene expression failure non-human animal can be used as a model for diseases caused by the failure of LLPL bioactivities, since various bioactivities capable of being derived from LLPL are deleted. Thereby, the LLPL gene expression failure non-human animal is useful for screening medicines for preventing and/or treating various diseases caused by the LLPL deficit, and for investigating the causes of the LLPL-related diseases and examining methods for treating the LLPL-related diseases. By the screening method, the medicines for preventing and/or treating the various diseases caused by the LLPL deficit can efficiently be screened. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a non-human mammalian embryonic stem cell in which an endogenous gene of lecithin: cholesterol acyltransferase-like lysophospholipase (LCAT-like lysophospholipase, sometimes abbreviated as LLPL hereinafter) is inactivated. , A non-human mammal deficient in LLPL gene expression, a method for screening a preventive and / or therapeutic drug using them, and a preventive and / or therapeutic drug obtainable by the screening.

[0002]

2. Description of the Related Art Advances in genetic engineering technology and rapid accumulation of knowledge of the underlying molecular biology have made it possible to artificially manipulate genes and further introduce them into individual animals [Gordon, JW Proc. Natl. Acad. Sci. USA, Proceedings of National Academy of Science USA, Vol. 77, 7
380-7384 (1980)]. For example, a method has been developed that artificially adds an exogenous genetic trait that is not originally present in the organism, or suppresses the expression of an endogenous genetic trait possessed by the organism. Animals into which various genetic traits have been introduced have been produced, and reported as transgenic animals or knockout animals. In order to clarify the functions of various cloned genes isolated by such techniques as genetic engineering, such transgenic animals have so far used in vitro cultured cells such as cell lines and primary cultured cells. Therefore, it is important to enable functional studies of genes, for which limited knowledge has been obtained, at the individual level. In particular, experiments and studies using this transgenic animal as an analysis of physiological functions of cloned genes in living organisms and as a model system for genetic diseases have become popular.

Embryonic stem cell:
ES cell) is a cell line established from an inner cell mass existing inside the blastocyst, which is an early embryo after fertilization, and is capable of proliferating and culturing while maintaining an undifferentiated state. These cells have pluripotency capable of differentiating into all kinds of cells in the living body, and when injected into normal early embryos, they can participate in the formation of embryo bodies and form chimeric animals. (Evans MJ and Kaufman MH, Nature (N
ature), Vol. 292, p. 154 (1981)]. Utilizing this property, attempts have been made to produce various genetically modified animals. Its history begins with the establishment of ES cells by Evans and Kaufman in 1981, and the production of ES chimeric mice by Bradley et al. [Nature, Vol. 309, page 255 (1984)].
The research has started in earnest. In addition Thomas and Capecchi
Homologous recombination of ES cells by [Cell, Vol. 51,
503 (1987)], the success of Germ line transmission of ES cell traits by three groups including the group of Koller et al. [Proc. Natl.
Acad. Sci. USA), Vol. 86, p. 8927 (1989)], and the rapid progress of research such as the production of gene-deficient mice. The ES cells that have been reported to date are Evan.
S. and Kaufman's EK cells [Id.], Doetschm
ES-D3 cells of an [J. Embryol. Exp. Molph., Vol. 87, p. 27 (198).
1)], Robertson's CCE cells [Nature,
Vol. 323, p. 445 (1986)], BL by Ledermann and Burki.
/ 6III cells [Experimental cell research (E
xp. Cell Res.), Vol. 197, p. 254], etc., but most of these were established from 129 strain mice.

Examples of gene expression-deficient animals produced by using such ES cells include (1) Hooper et al. [Nature, Vol. 326, pp. 292 (1987)] and Knehn et al. [Nature (Nature)]. ), 326, 295 (1987)], the HPRT gene-deficient mouse using spontaneous mutant ES cells, (2) Donehower et al. [Nature, 356].
, 215 (1992)], one of the tumor suppressor genes, p5
P3 deletion mouse lacking 3 and (3) β2 of Zijlstra et al. [Nature, 344, 742 (1990)].
Microglobulin gene mutant mouse, (4) Sinkai et al., Which is one of immune disease model mice [Cell, 68th
Vol. 855 (1992)] RAG-2 (V (D) Jrecombinati
on activation gene) mutant mouse, (5) Glimcher et al. [Science, Volume 253, page 1417 (1991)
)] And Cosgrove et al. [Cell, Vol. 66, page 1051]
(1991)], MHC class II mutant mouse, and (6) development / development-related disease model mouse as one of MacMahon et al. [Cell, Volume 62, 1073 (1990)] int
-1 gene deficient mouse and (7) Soriano et al. [Cel (Cel
l), Vol. 64, p. 693 (1991)], src-deficient mice exhibiting marble disease-like symptoms have been reported.

On the other hand, for lipoprotein metabolism, apoprotein [Srivastava, R. & A., Srivastava, N. (2000) Mol.
Cell Biochem., 209, 131-144], enzymes, receptors [Hil
tunen, T., P. & Yla-Herttuala, S. (1998) Atheroscl
erosis, 137, S81-88], lipid transfer protein [Yamashit
a, S. et al, (2000) Atherosclerosis, 152, 271-285, Or
am, J., F. & Vaughan, A., M. (2000) Curr. Opin. Li
pidol., 11, 253-260], but individual differences caused by different genotypes of these proteins and different diets are closely related to arteriosclerosis.
One of them, high-density lipoprotein (HDL) cholesterol, has been recognized by many epidemiological studies as an independent negative risk factor for coronary artery disease [Barter,
P., J. & Rye, K., A. (1996) Atherosclerosis, 121,
1-12]. HDL plays a major role in the so-called reverse cholesterol transfer system, which extracts excess cholesterol from peripheral tissues and transfers it to the liver [Tall, A. et al.
(2000) Arterioscler.Thromb. Vasc. Biol., 20, 1185
-1188, Santamarina-Fojo, S. et al. (2000) Curr. Opin. L.
ipidol., 11, 267-75]. This rate-limiting enzyme, lecithin: cholesterol acyltransferase (LCA)
T) is mainly produced in the liver [Warden, C. et al., (198
9) J. Biol. Chem., 264, 21573-21581], H in blood
It is present in DL particles [Kuivenhoven, J. et al., (199
7) J. Lipid Res., 38, 191-205]. Peripheral cell free cholesterol extracted by PreβHDL is
Since it is esterified by LCAT and the influx of cholesterol into cells is no longer suppressed, it eventually shows the action of promoting cholesterol withdrawal, and is considered to have an anti-atherosclerotic effect [Czarnecka, H. & Yokoyama. ,
S. (1995) Biochemistry, 34, 4385-92]. Recently, the enzyme LLPL, which has 47% amino acid homology to human LCAT, has been detected in human macrophage-like cell cD.
It was discovered by a subtraction PCR method using an NA library, and its entire amino acid sequence has been clarified by analysis of cDNA [Taniyama et al .; Biochemical and Biophysical Research Communications (Biochem. Biophys. Res. Commun .), 257: 50-56
(1999)]. Although a large amount of foamed macrophages are accumulated in human arteriosclerotic lesions, LLPL is secreted by human macrophage-like cells, and ApoE (apolipoprotein E) is one of the genes related to arteriosclerosis. ) Is known, but in situ hybrid in arteriosclerotic lesions of arteriosclerosis model mouse
Since the expression of the mouse LLPL gene in the lesion was confirmed from the results of ization, it is assumed that the LLPL gene is closely related to the progression of arteriosclerosis. Human L
LPL esterifies free cholesterol LCAT
It has no activity, but shows lysophospholipase activity that decomposes into free fatty acids and glycerophosphorylcholine using lysophosphatidylcholine as a substrate in vitro [Taniya
ma et al .; Biochemical and Biophysical Research Communications (Biochem. Biophys. Re
s. Commun.), 257: 50-56 (1999)]. It has also been confirmed that this human LLPL exists in human serum, suggesting the presence of a substrate of unknown identification in blood. LLPL is secreted by macrophage-like cells in humans and peritoneal macrophages in mice, and there is 88% homology in amino acid sequence between humans and mice,
It is known that the lipase motif has the same AHSMG sequence (Japanese Patent Laid-Open No. 11-269199), and there are many similarities in that the expression specificity of the LLPL gene in the whole individual is in peripheral tissues. Furthermore, since LLPL has been confirmed to be expressed in atherosclerotic lesions of ApoE-deficient mice, it may have an effect on arteriosclerotic lesions and plasma lipid profile. However, these actions have not been fully elucidated, and many points remain to be elucidated regarding the function and action mechanism of LLPL in vivo. LLP
In elucidating the action of L in the living body, LLPL
Although a model of an LLPL gene expression-deficient animal that produces no or only a small amount of L.A. has been craved as an essential one, such an animal model has not yet been created.

[0006]

Therefore, the LLPL
If a non-human animal ES cell in which a gene is inactivated is successfully produced, a non-human animal deficient in LLPL gene expression can be produced. Since the obtained non-human animal deficient in LLPL gene expression lacks various biological activities that can be induced by LLPL, it serves as a model of diseases caused by inactivation of biological activity of LLPL, and causes these diseases. It becomes possible to investigate and study treatment methods.

[0007]

Means for Solving the Problems In view of the above-mentioned problems, the present inventors have conducted extensive studies and as a result, succeeded in producing a non-human animal lacking LLPL gene expression, and as a result of further research, The present invention has been completed. That is, the present invention provides (1) a mammalian embryonic stem cell in which a lecithin: cholesterol acyltransferase-like lysophospholipase gene is inactivated; (2) an embryonic stem cell of the above (1) which is drug resistant; (3) a neomycin drug. The embryonic stem cell according to (2) above; (4) The embryonic stem cell according to (1) above, wherein the lecithin: cholesterol acyltransferase-like lysophospholipase gene is inactivated by insertion of the reporter gene; (5) the reporter gene is the lacZ gene. (6) The embryonic stem cells of (4) above; (6) The embryonic stem cells of (1) above, wherein the mammal is a rodent; (7) The embryonic stem cells of (6) above, wherein the rodent is a mouse; (8) Lecithin: Non-human mammal deficient in expression of cholesterol acyltransferase-like lysophospholipase gene or its tissue Is a cell derived from them; (9) the animal of the above (8) or a tissue thereof derived from the non-human mammal is a rodent, or a cell derived therefrom; (10) the animal of the above (8) or a tissue thereof, or them. For preventing diseases caused by deficiency of lecithin: cholesterol acyltransferase-like lysophospholipase gene, characterized by using cells derived from
Or a method for screening a therapeutic agent; (11) Due to a deficiency of lecithin: cholesterol acyltransferase-like lysophospholipase gene, which comprises administering a test compound to the animal or the tissue thereof or the cells derived therefrom in (8) above. A method for screening a preventive and / or therapeutic agent for a disease, wherein: (12) the screening method according to (10) or (11) above, wherein the disease is arteriosclerosis; (13) the screening method according to (10) or (11) above The resulting preventive and / or therapeutic agent for a disease caused by a deficiency of a lecithin: cholesterol acyltransferase-like lysophospholipase gene; (14) the preventive and / or therapeutic agent for the above (13), wherein the disease is arteriosclerosis; (15) By mating the animal of (8) above with other pathological model animals (16) A disease model animal or a tissue thereof or a cell derived from the disease model animal or the tissue thereof, which is caused by drug induction or stress load on the animal of (8) above; (17) Other (15) The disease model animal of the above (15), wherein the disease model animal is a non-human mammal deficient in apolipoprotein gene expression, or cells derived therefrom; (18) the mammal is a lecithin: cholesterol acyltransferase-like lysophospholipase gene and (17) The pathological condition model animal of the above-mentioned (17) which is a non-human mammal deficient in the apolipoprotein gene, or a cell derived therefrom; (19) The above-mentioned (17) pathological model animal in which the apolipoprotein is apolipoprotein E If (20) The tissue or cells derived therefrom; (20) The animal model of the disease state caused by drug induction or stress load to the animal of (15) above, the tissue thereof or cells derived therefrom; (21) (15) to (17) above Or a screen for a preventive and / or therapeutic agent for a disease caused by a deficiency of a lecithin: cholesterol acyltransferase-like lysophospholipase gene, which comprises using the animal of any one of (20) or a tissue thereof, or a cell derived therefrom. Method: (22) Lecithin: cholesterol acyltransferase-like lyso, which comprises administering a test compound to the animal or tissue thereof or cells derived therefrom according to any of (15) to (17) or (20) above. Caused by a deficiency in the phospholipase gene Prevention of diseases and /
Or (23) a preventive and / or therapeutic agent for a disease caused by a deficiency of lecithin: cholesterol acyltransferase-like lysophospholipase gene obtained by the screening method of (22) or (23) above; (24) Prevention of a disease caused by a deficiency of lecithin: cholesterol acyltransferase-like lysophospholipase gene, which comprises using the animal of the above (17) or a tissue thereof or cells derived from the tissue and / or
Or a method for screening a therapeutic agent; (25) Due to a deficiency of a lecithin: cholesterol acyltransferase-like lysophospholipase gene, which comprises administering a test compound to the animal or the tissue thereof or the cells derived from the animal of (17) above. (26) A method for screening a preventive and / or therapeutic drug for a disease: (26) Preventing and / or preventing a disease caused by a deficiency in a lecithin: cholesterol acyltransferase-like lysophospholipase gene obtained by the screening method according to (24) or (25) above. To provide a remedy.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION [LLPL Gene-Inactivated Mammalian ES Cell] A mammalian ES cell in which the LLPL gene is inactivated is an endogenous LLPL possessed by the mammalian ES cell.
By artificially mutating a gene, the expression ability of the gene is suppressed, or the activity of the LLPL encoded by the gene is substantially lost, so that the gene substantially expresses the LLPL. It refers to a mammalian ES cell that is inactivated so that it does not have (hereinafter, may be referred to as the knockout gene of the present invention). In this specification,
Examples of mammals used as a material for ES cells include humans, cows, pigs, sheep, goats, rabbits, dogs, cats,
Guinea pigs, hamsters, mice, rats, etc. are used. In the present specification, non-human animals include LLP.
Any animal other than human having the L gene may be used, but a non-human mammal is preferable. Examples of the non-human mammal include cow, pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat and the like. Among non-human mammals, rodents, especially mice (for example, C57BL / 6 strain as a pure strain, which have relatively short ontogenesis and biological cycle from the viewpoint of preparation of pathological animal model system, and are easy to reproduce)
As a hybrid system such as DBA2 strain, B6C3F1 strain,
BDF1 system, B6D2F1 system, BALB / c system,
ICR system, etc. (among others, preferably as a pure system, C
57BL / 6 strain, such as BDF1 strain or ICR strain) or rat (eg, Wis)
tar, SD, etc.) are particularly preferable.

The method for artificially mutating the LLPL gene includes, for example, deletion of a part or all of the gene sequence or insertion or substitution of another gene by a genetic engineering technique. Due to these mutations, for example,
The knockout gene of the present invention can be prepared by shifting the reading frame of the codon or disrupting the function of the promoter or exon. In order to prepare a mammalian (preferably non-human mammal) ES cell in which the LLPL gene is inactivated (hereinafter abbreviated as LLPL gene inactivated ES cell or knockout ES cell), specifically, For example, drug resistance genes (eg,
Neomycin resistance gene, hygromycin resistance gene, zeocin resistance gene, etc., preferably neomycin resistance gene, etc., or reporter gene (eg, lacZ (Escherichia coli β-galactosidase gene), cat (chloramphenicol acetyltransferase gene), GUS) (Β-glucuronidase gene), luciferase gene, aequorin gene, taumarin gene, GFP (Green Fluorescent Protei)
n) A gene or the like, preferably lacZ or the like) is inserted to disrupt the function of the exon of the LLPL gene, or a DNA sequence (eg, polyA addition signal etc.) that terminates the transcription of the gene in the intron portion between the exons. ) Is inserted to make it impossible to synthesize complete mRNA, resulting in a DNA vector having a DNA sequence constructed so as to disrupt the gene (hereinafter abbreviated as targeting vector). When a reporter gene is inserted to disrupt the function of exon, the reporter gene is preferably inserted so as to be expressed under the control of the LLPL promoter. The "drug resistance gene" refers to a gene involved in drug resistance such as antibiotics, and is used as a marker for selecting whether or not the introduced gene is expressed in cells. Further, the "reporter gene" refers to a group of genes that serve as an index of gene expression, and usually a structural gene of an enzyme that catalyzes a luminescence reaction or a color reaction is used,
(1) No genetic background, (2) High-sensitivity method for quantitative gene expression, (3) Little effect on transformed cells, (4) Localization of expression site And the like are preferably used (plant cell engineering,
Volume 2, p. 721, 1990). Further, the above-mentioned “drug resistance gene” and the like are also used for the same purpose, but the “reporter gene” not only checks whether or not the introduced gene is expressed in cells but also in which tissue and when it is expressed. In addition, the expression level can be accurately examined quantitatively. Next, the above targeting vector is introduced into the chromosome of the ES cell by, for example, a homologous recombination method, and the obtained ES cell is subjected to Southern hybridization analysis or targeting vector using a DNA sequence on or near the LLPL gene as a probe. It can be obtained by selecting the knockout ES cells of the present invention by analysis by the PCR method using the above DNA sequence and the DNA sequence in the vicinity region other than the LLPL gene used for producing the targeting vector as a primer. Examples of the above-mentioned targeting vector include plasmids derived from E. coli (eg, pBR322, pB
R325, pUC12, pUC13, etc.), a plasmid derived from Bacillus subtilis (eg, pUB110, pTB5, pC19)
4, etc.), yeast-derived plasmids (eg, pSH19, p
SH15 etc.), bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, vaccinia virus or adenovirus vector, baculovirus, bovine papilloma virus, viruses from the herpesvirus group, or animals such as Epstein-Barr virus. A virus or the like is used.

In addition, the endogenous LLPL can be prepared by the homologous recombination method or the like.
As an original ES cell for inactivating a gene, for example, an already established one as described above may be used,
It may be newly established according to the known method of Evans and Kaufman. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used. However, since the immunological background is not clear, an alternative pure line immunological genetic background is used. For the purpose of obtaining ES cells whose C.P.
Those established by using F1) with A / 2 can also be favorably used. The BDF1 mouse has a large number of eggs and has the advantage that the eggs are strong, and since it has C57BL / 6 mouse as a genetic background, the ES cells obtained by using it produced pathological model mice. When C57BL / 6
The genetic background of C57B was improved by backcrossing with mice.
It can be advantageously used in that it can be replaced with an L / 6 mouse. When ES cells are established, blastocysts on the 3.5th day after fertilization are generally used, but other than this, 8-cell stage embryos (8-cell stage embryos around 2.5 days after fertilization are preferred. (1) can be collected efficiently, and a large number of early embryos can be efficiently obtained.

The second screening can be carried out by confirming the number of chromosomes by the G-banding method. It is desirable that the number of chromosomes of ES cells obtained is 100% of the normal number, but if it is difficult due to physical manipulation during establishment, after knocking out the gene of ES cells, normal cells (for example, the number of chromosomes in mice are Is preferably 2n = 40). The ES cell line thus obtained usually has a very good proliferative property, but since it is easy to lose the ability of ontogeny development, careful subculture is necessary. For example, LIF (1-1) on suitable feeder cells such as STO fibroblasts.
Culturing in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5% carbon dioxide, 90% air) in the presence of 0000 U / ml at about 37 ° C. However, at the time of subculture, for example, trypsin / EDTA
Solution (usually 0.001-0.5% trypsin / 0.1-5
mM EDTA, preferably about 0.1% trypsin / 1m
(M EDTA) treatment to obtain single cells and seeding on newly prepared feeder cells. Such subculture is usually performed every 1-3 days, but at this time, if cells are observed and morphologically abnormal cells are found, it is desirable to discard the cultured cells. ES cells are differentiated into various types of cells such as parietal muscle, visceral muscle, and myocardium by performing monolayer culture until reaching a high density or suspension culture until forming a cell clump under appropriate conditions. (MJ Evans and M.
H. Kaufman, Nature, Volume 292, 154, 19
1981; GR Martin Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1
981; TC Doetschman et al., Journal of Embryology and Experimental Morphology, Vol. 87, p. 27, 1985], LLPL gene expression-deficient cells obtained by differentiating ES cells of the present invention , In
It is useful in the cell biological study of LLPL in vitro. When ES cells are stored, a suitable freezing medium (for example, Dulbecco's modified Eagle medium (DMEM) containing 10% DMSO and 10% fetal bovine serum) is used at about -80 ° C or lower. Store frozen.

[LLPL gene expression-deficient non-human animal] The LLPL gene expression-deficient non-human animal of the present invention (hereinafter sometimes referred to as gene expression-deficient non-human animal) is, for example,
The LLPL gene is produced by genetic engineering using cells derived from mammalian ES cells in which the LLPL gene is inactivated, and, for example, germline and somatic cells are provided with an inactivated LLPL gene sequence in the early stage of embryogenesis. It is an introduced non-human animal. As the non-human animal, the same ones as described above are used. In order to knock out the LLPL gene, the above-mentioned targeting vector can be used for non-human animal ES cells or non-human animal egg cells by known methods (for example, electroporation method, microinjection method, calcium phosphate method, lipofection method, agglutination method, particle gun). Method, DEAE-dextran method, etc.) (a preferable introduction method includes an electroporation method when introducing into ES cells and a microinjection method when introducing into egg cells),
It can be carried out by replacing the inactivated LLPL gene sequence of the targeting vector with the LLPL gene on the chromosome of the non-human animal ES cell or the non-human animal egg cell by homologous recombination. Cells in which the LLPL gene was knocked out were analyzed by Southern hybridization analysis using a DNA sequence on or near the LLPL gene as a probe or D on the targeting vector.
It can be determined by analysis by the PCR method using the NA sequence and the DNA sequence in the vicinity region other than the mouse-derived LLPL gene used as the targeting vector as primers. When non-human animal ES cells are used, a cell line in which the endogenous LLPL gene is inactivated is cloned by homologous recombination, and the cells are cloned at an appropriate stage in the early stage of embryogenesis, for example, at the 8-cell stage A chimeric embryo prepared by injecting into a human animal embryo or blastocyst (injection method) or sandwiching an ES cell mass in which the LLPL gene is inactivated with two 8-cell stage embryos (collective chimera method) The pseudopregnant non-human animal is transplanted into the uterus. The animal thus produced is a chimeric animal composed of both cells having a normal LLPL locus and cells having an artificially mutated LLPL locus. When a part of the germ cells of the chimeric animal has a mutated LLPL locus, all tissues are artificially mutated from the population obtained by mating such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the LLPL locus, for example, by judging the coat color. The individual thus obtained is usually an LLPL hetero expression deficient individual, and LLPL hetero expression deficient individuals can be crossed to obtain LLPL homo expression deficient individuals from their offspring. When using egg cells, for example,
By injecting the targeting vector solution into the nucleus of the egg cell by the microinjection method, a transgenic non-human animal in which the gene is introduced into the chromosome can be obtained, and by comparing these transgenic non-human animals, homologous recombination is performed. It is obtained by selecting those having a mutation in the LLPL locus. LLP
A non-human animal deficient in L gene expression can be distinguished from a normal animal by measuring the mRNA amount of the animal using a known method and indirectly comparing the expression amount thereof.

As described above, the individual in which the LLPL gene has been knocked out can be passaged in a normal breeding environment after confirming that the gene has been knocked out in the animal individuals obtained by mating. . further,
The germ line can be obtained and maintained according to a conventional method. That is, by mating male and female animals having the inactivating gene sequence, a homozygous animal having the inactivating gene sequence on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by raising the mother animal in such a state that there are 1 normal individual and 1 homozygous animal. By mating male and female heterozygous animals, the homozygous and heterozygous animals having the inactivated gene sequence can be passaged. The offspring of the thus obtained animal having the inactivated gene sequence is also included in the LLPL gene expression-deficient non-human animal of the present invention. [Evaluation of mammals deficient in LLPL gene expression] LL of the present invention
Non-human animal deficient in PL gene expression (particularly, LLPL homo-deficient non-human animal, preferably LLPL homo-deficient mouse)
Has the following properties. (1) When fed a high-fat high-cholesterol diet, total cholesterol and LDL + VLDL cholesterol in plasma, triglyceride in the liver, phospholipids,
Free cholesterol, total cholesterol, and liver weight tend to be lower than those of wild-type animals. (2) By giving a high fat and high cholesterol diet,
On histopathological examination, fatty liver findings such as increased vacuoles and disordered cord structures due to excessive fat accumulation in hepatocytes, increased single cell necrosis, large and small nuclei, and mononuclear cell infiltration were observed.
Mild compared to wild type animals. (3) By giving a high fat and high cholesterol diet,
Among the lipid contents of the liver, triglyceride, phospholipid, and free cholesterol are significantly lower than those of wild-type animals. The LLPL / ApoE double-deficient non-human animal (especially mouse) of the present invention has the following properties. (1) The area ratio of arteriosclerotic lesions is increased by about 2 to 3 times as compared with ApoE-deficient animals. [Pathological Model Animal] Mammalian ES cells in which the LLPL gene is inactivated in this manner are very useful for producing a non-human animal deficient in expressing the LLPL gene. Also, L
LPL gene expression deficient non-human animal, pathological model animal caused by drug induction or stress load to the animal, better pathological model animal caused by mating of LLPL gene expression deficient non-human animal with other pathological model animal, LLPL gene expression deficiency A bone marrow transplant animal using a disease state model animal caused by drug induction or stress load on a disease state model animal produced by mating a non-human animal with another disease state model animal, and a bone marrow transplant animal using a non-human animal LLPL gene expression deficient non-human animal and another disease state model animal, or Those tissues or cells derived from them are associated with diseases caused by LLPL deficiency, for example, diseases caused by inactivation of LLPL biological activity based on the loss of various biological activities that can be induced by LLPL (for example, , Arteriosclerosis, hypercholesterolemia, high Lyseridiaemia, hyperlipidemia, diabetic complications, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, insulin-dependent diabetes mellitus, liver disorder, adrenal insufficiency, senile pancreatic hypofunction, etc., preferably Can be a better model system for arteriosclerosis, etc., and is useful for investigating the causes of these diseases and investigating treatment methods. Here, other pathological model animals include, for example, arteriosclerosis model mice such as LDL receptor-deficient mice, ApoE-deficient mice, CETP-introduced mice, ApoB-introduced mice and the like, or mice obtained by mating thereof, or impaired glucose tolerance or Insulin resistance or both
Type II diabetes model mouse (KK mouse, KKA ^y mouse, ob / o
b mouse, db / db mouse, IRS-1 deficient mouse, IRS-2 deficient mouse, Glucokinase deficient mouse and the like or mice obtained by crossing thereof) and type I diabetes model mouse (NOD mouse and the like). In addition, a model mouse by bone marrow transplantation in which the gene expression defect or increase is localized only in the blood system is also included [Linton, MF, et al.
al., Science 267: 1034-1037 (1995)]. Bone marrow transplanted mice have the potential of becoming more suitable pathological model animals because the site of altered gene function is localized. For example, as a donor animal, a pathological model animal obtained from the above-described non-human animal lacking LLPL gene expression, its bone marrow was collected, and the mouse was transplanted to another recipient animal whose bone marrow was destroyed by irradiation in advance, or Using other pathological model animals as donor animals, collecting the bone marrow,
It also includes a bone marrow transplant mouse in which a pathological model animal obtained from a non-human animal deficient in LLPL gene expression in which bone marrow was destroyed by irradiation in advance was transplanted as a recipient animal. For example, in the case of arteriosclerosis, in the former, macrophages infiltrating the lesion do not express LLPL, and in the latter, only macrophages infiltrating the lesion express LLPL. According to a preferred embodiment of the present invention, other pathological model animals that are bred with LLPL gene expression-deficient non-human animals include, for example, ApoE gene-deficient animals associated with arteriosclerosis. LL of the present invention
In a dual-function deficient model animal produced by mating a PL-deficient non-human animal and an arteriosclerosis model animal, the relationship with a disease caused by arteriosclerosis-related gene ApoE, prevention of various diseases caused by LLPL deficiency and ApoE deficiency, and / or Alternatively, it is useful for screening therapeutic agents, investigating the cause of LLPL- and ApoE-related diseases, and examining the related therapeutic methods. Thus, the LLPL gene expression-deficient non-human animal of the present invention, the pathological model animal caused by drug induction or stress load on the animal, and the pathological model animal produced by mating of the LLPL gene expression-deficient non-human animal with other pathological model animals , LLPL gene expression-deficient non-human animals and other pathological model animals caused by drug induction or stress load on the pathological model animals, pathological model animals obtained from LLPL gene-deficient non-human animals and others The disease state model animal obtained by bone marrow transplantation using the disease state model animal of, or a tissue thereof or cells derived therefrom can be used for screening of a preventive and / or therapeutic drug for the disease. Here, the tissue or cells derived therefrom are used for screening by measuring a specific activity using a homogenate such as liver or kidney, or measuring the activity or production amount of a specific product using peritoneal macrophages. Can be used.

[Screening Method of the Present Invention] A non-human animal lacking LLPL gene expression used in the screening method of the present invention, a disease state model animal caused by drug induction or stress load of the animal, non-human animal lacking LLPL gene expression and other animals Disease model animal caused by mating with disease model animal, LLPL gene expression deficiency Disease model animal caused by drug induction or stress load on disease model animal caused by mating of non-human animal with other disease model animal, LLPL gene expression failure A pathological model animal obtained by bone marrow transplantation using a pathological model animal obtained from a human animal and another pathological model animal,
Alternatively, examples of those tissues or cells derived therefrom include the same ones as described above. Examples of test compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. Even if these compounds are novel compounds It may be a known compound. Specifically, a non-human animal deficient in LLPL gene expression of the present invention, a pathological model animal produced by drug induction or stress load of the animal, a pathological model produced by mating of a non-human animal deficient LLPL gene expression with another pathological model animal Animal, a disease state model animal obtained by crossing of a non-human animal lacking LLPL gene expression with another disease state model animal caused by drug induction or stress load to a disease state model animal, and a disease state model animal obtained from a non-human animal lacking LLPL gene expression A test compound is used as a test compound for a disease state model animal obtained by bone marrow transplantation using another disease state model animal (hereinafter, may be referred to as non-human animal with LLPL gene expression deficiency), or a tissue thereof or cells derived from them. Treated with, and compared to untreated control animals, , It is possible to test cell, the preventive and therapeutic effect of the test compound as an indicator of changes such symptoms of the disease. As a method for treating a test animal (a non-human animal deficient in LLPL gene expression, etc.) with a test compound, for example, oral administration, intravenous injection, etc. are used, and it is appropriately selected depending on the symptoms of the test animal, the properties of the test compound, etc. be able to. Further, the dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like. For example, in the case of screening a preventive and / or therapeutic drug for arteriosclerosis or hyperlipidemia, the LLPL gene expression-deficient non-human animal of the present invention is subjected to cholesterol loading treatment, and the test compound is administered before or after the cholesterol loading treatment. After administration, the blood cholesterol level and body weight change of the animal can be measured over time to perform screening. When screening a preventive and / or therapeutic drug for type I diabetes, a non-human animal deficient in expressing the LLPL gene of the present invention is administered with a drug such as STZ or alloxan, and a test compound is administered before or after glucose tolerance treatment. However, it can be screened by measuring the blood glucose level and weight change of the animal over time.

[Preventive and / or therapeutic agent obtained by the screening method of the present invention] The prophylactic and / or therapeutic agent obtained by the screening method of the present invention contains a compound selected from the above-mentioned test compounds And
Since it has a preventive / curative effect on a disease caused by LLPL deficiency, it is useful as a medicine such as a safe and low-toxic therapeutic / prophylactic drug against a disease caused by LLPL deficiency. Further, a compound derived from the compound can be used as well. The compound obtained by the screening method may form a salt, and examples of the salt of the compound include a salt with an inorganic base, a salt with an organic base, a salt with an inorganic acid, and a salt with an organic acid. , Pharmaceutically acceptable salts such as salts with basic or acidic amino acids, and the like. Preferable examples of the salt with an inorganic base include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and aluminum salt and ammonium salt. Suitable examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline,
2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine,
Examples thereof include salts with dicyclohexylamine and N, N'-dibenzylethylenediamine. Preferable examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and the like. Preferable examples of salts with organic acids include, for example, formic acid, acetic acid, propionic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid. And salt. Preferable examples of the salt with basic amino acid include salts with arginine, lysine, ortinine, and preferable examples with the acidic amino acid include salts with aspartic acid, glutamic acid and the like.

When the compound or its salt obtained by using the screening method of the present invention is used as the above-mentioned therapeutic / prophylactic agent, it can be carried out according to a conventional method, for example, a tablet coated with sugar if necessary, Orally as capsules, elixirs, microcapsules, etc.
Alternatively, it can be used parenterally in the form of an injectable solution such as a sterile solution or suspension with water or other pharmaceutically acceptable liquid. For example, the compound or a salt thereof is mixed with a pharmaceutically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like in a unit dose form generally required for pharmaceutical practice. It can be manufactured by The amount of active ingredient in these preparations is such that a suitable amount within the indicated range can be obtained. Additives that can be mixed with tablets, capsules and the like include, for example, binders such as gelatin, corn starch, tragacanth, and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Puffing agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry and the like are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like.

Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride) and the like. Also used in combination with an auxiliary agent such as alcohol (eg ethanol), polyalcohol (eg propylene glycol, polyethylene glycol etc.), nonionic surfactant (eg Polysorbate 80 ^™ , HCO-50 etc.) and the like. Good. Examples of the oily liquid include sesame oil, soybean oil and the like, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer, etc.), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), Preservatives (eg,
(Benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, for example, humans or warm-blooded animals (for example, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.) ). The dose of the compound or its salt varies depending on the symptoms, but in the case of oral administration, it is generally about 0.1 to 100 m per day in an adult (with a body weight of 60 kg) arteriosclerosis patient.
g, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally,
Although the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., for example, in the form of an injection, in an adult (60 kg) arteriosclerosis patient, the daily dose is about 0.01 to about About 30 mg, preferably about 0.
1 to 20 mg, more preferably about 0.1 to 10 mg
It is convenient to administer the degree intravenously. Also in the case of other animals, the dose converted per 60 kg of body weight can be administered.

In the present specification and drawings, when abbreviations are used for bases, amino acids, etc., IUPAC-IUB
Based on the abbreviations used in Commision on Biochemical Nomenclature or conventional abbreviations in this field,
An example is given below. When amino acids may have optical isomers, the L form is shown unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid Gly: glycine Ala: alanine Val: valine Leu: leucine Ile: isoleucine Ser: serine Thr : Threonine Cys: Cysteine Met: Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glut:

The sequence numbers in the sequence listing in this specification show the following sequences. [SEQ ID NO: 1] This shows the amino acid sequence of mouse-derived LLPL. [SEQ ID NO: 2] DN encoding mouse-derived LLPL
The base sequence of A is shown. [SEQ ID NO: 3] This shows the base sequence of primer P94-1. [SEQ ID NO: 4] This shows the base sequence of primer P101-4. [SEQ ID NO: 5] This shows the base sequence of primer SI-75. [SEQ ID NO: 6] This shows the base sequence of primer P3-t. [SEQ ID NO: 7] This shows the base sequence of primer P4-s.

[0020]

EXAMPLES The present invention will be described in more detail below with reference to Reference Examples and Examples, but the scope of the present invention is not limited thereto.

Reference Example 1 Culture of ES cells AB2.2-Prime ES Cells derived from 129 / SvEv as ES cells
(LEXICON) using the method of Joyner et al. (Gene Targeting:
A Practical Approach, 1993, Oxford University Pre
The culture was performed according to ss or its translation, Gene Targeting, Tetsuo Noda, Medical Science International Co., Ltd.). ES cells and feeder cells are recommended 15% FBS, 2 mM L-Glutamin
e, 10 ^-4 M β-Mercaptoethanol, 50U / ml Penicillin, 50
Using ESQ DMEM medium (medium for ES cells) containing μg / ml Streptomycin, the cells were cultured at 37 ° C. in the presence of 5% CO ₂ . ES
The cells were cultured on a feeder cell layer that had been arrested by culturing in a medium supplemented with 10 μg / ml Mitomycin C (Sigma) for 3 hours. In order to suppress the differentiation of ES cells as much as possible, the medium exchange was basically a half volume exchange, and the cells were passaged every 2-3 days.

Reference Example 2 Screening of mouse genomic DNA library pCMV-SPORT plasmid (pTB2010) containing mLLPL (mouse LLPL) cDNA was treated with SmaI restriction enzyme to obtain mLLPL from MCS (multi cloning site) of the vector located at the N-terminal. About 1 kbp up to the SmaI site was isolated. Using this as probe A, the BAC ES Mouse Hybridization library screening from the 129 / Svj mouse genome library
mLLPL using services (GENOME SYSTEMS)
MLLPL / pBeloBac1 containing the entire genomic sequence of
Three clones of 1 were obtained. About these 3 clones, Eco
MLLPL ORF (open reading frame) for DNA fragments fragmented by RI and PstI restriction enzyme treatments
Was analyzed by the Southern hybridization method using a DNA probe including almost the entire region. The DNA probe is a sense strand primer P using pTB2010 as a template.
94-1 [5'-GGT AAC CAG TTG GAA GCA AAG-3 '; 21 mers
(SEQ ID NO: 3)] and antisense strand primer P101-
4 [5'-CCC CGG GTG GCG TCA T-3 '; 16 mers (SEQ ID NO:
4)] was used for the PCR method. From the EcoRI-treated product, two DNA fragments of about 11 kbp and about 7.5 kbp were
About 6.5 kbp, about 1.5 kbp and about 0.6 kb from stI-treated products
Three DNA fragments of p were obtained as positive bands, and a total of 5 types of mLLPL positive bands could be detected. Each of these was subcloned into the pUC118 vector to obtain plasmids EcoRI (11 kbp) / pUC118, EcoR
I (7.5 kbp) / pUC118, PstI (6.5 kbp) / pUC118, PstI (1.5
kbp) / pUC118 and PstI (0.6 kbp) / pUC118 were obtained, and restriction enzyme mapping and nucleotide sequence were analyzed. It was revealed that the obtained clone covers about 13 kbp of the entire region of the mLLPL genome, and 13 kbp mLLPL
The exon-intron structure on the genome was determined (Fig. 1). The ORF of mouse LLPL is composed of a total of 6 exons, and it was revealed that the lipase motif predicted to be the active site thereof exists on the 5th exon.

Reference Example 3 Construction of Targeting Vector pTB2224 Using the plasmid obtained by fragmenting mLLPL / pBeloBac11 by restriction enzyme treatment and subcloning into pUC118 vector, short arm and long arm DNA fragments were prepared. HindIII / PstI fragment containing exon 6 (1.1 kb
EcoRI fragment containing the first and second exons (11 kbp) using short arm with SalI site added to both ends of p).
The following operations were performed in order to use the ones with NotI sites added to both ends as long arms. EcoRI (7.5 kbp) / pUC11
Isolate the HindIII fragment (1.3kbp) from 8 plasmids to obtain pBlu
A clone in which the 5'-end of the MCS insert was near the SalI site was selected by cloning into the HindIII site of escriptII. After cutting this plasmid with BamHI, T4 DNA poly
The ends were blunted using merase to obtain a plasmid having SalI linker inserted. This is digested with PstI and then self-cyclized to insert the PstI fragment (0.2
kbp) was excluded. This was cleaved with SalI to obtain a short arm DNA fragment with SalI sites added to both ends. On the other hand, an EcoRI (11 kbp) fragment cloned into the EcoRI site of pGEM-T Easy vector (Promega)
Lot with NotI sites added at both ends by cutting with NotI
A DNA fragment to be ng arm was obtained. Prepared short ar
m and long arm respectively with pPolII short-neobPA-HSVTK ve
ctor (Ishibashi S. et al., J. Clin. Invest. 1994; 93
(5): 1885-93) by ligation to the XhoI site and NotI site of the targeting vector pTB22.
24 were constructed (Figure 2).

Example 1 Selection of LLPL gene-deficient ES cells ES cells obtained in [Reference Example 1] that had grown to nearly confluency were treated with trypsin by the following method 3 hours after the medium was exchanged to obtain an ES cell suspension. Was prepared.
The ES cells (T-75) were washed 3 times with 10 ml of PBS / EDTA and added to 0.25
After suspending in 3 ml of a trypsin / 1 mM EDTA solution (Gibco BRL), 7 ml of D-PBS (−) was further added to suspend the cells, and the cells were collected by centrifugation. Collected cells in D-PBS (-)
After washing 3 times with 10 ml, resuspend in 1 ml of D-PBS (-) E
An S cell suspension was prepared. Targeting vector DNA (about 120 μg) pTB2224 obtained in [Reference Example 3]
Was linearized by SalI restriction enzyme treatment, extracted with phenol-chloroform, purified by ethanol precipitation and dissolved in 100 μl of Milli-Q water. DP in this DNA solution
0.9 ml of BS (-) was added to make 1 ml, and the suspension was added to the ES cell suspension prepared above and left for 3 minutes. Dispense 1 ml each into an electroporation cuvette and use Cell PoratorElectro
Electroporation into ES cells was performed using poration System I (Gibco BRL) (set to Charge and 330
After checking μF and Low Ω, raise the monitor value to about 300 with Fast-Up. Next, set to Arm and use Med-Down to monitor 2
When it reaches 75, quickly press Trigger). The cells after electroporation were transferred to 40 ml of a medium for ES cells, gently stirred, and then seeded on feeder cells arrested on four 10 cm dishes. 24 hours after seeding, G418 (final concentration 190 μg / ml: Gibco BRL) was added to the medium, and 1- (2-deoxy-2-fluoro-β-D-arabinofurano was added 4 days after electroporation. Sill) -5-iodouracil (FIAU; final concentration 100 ng / ml) was added to LL
Selective culture of PL gene-deficient ES cells was performed. The medium was exchanged every day, colonies grown on the 9th to 11th days were collected, and the target clones were selected by the primary screening by Southern analysis of the PCR product and the secondary screening by genomic Southern analysis. The LLPL gene deficiency of the obtained G418 resistant strain was determined by using the cell lysate prepared from a part of each collected colony as a template and the sense strand primer on the neomycin resistance gene.
P using SI-75 (SEQ ID NO: 5) and antisense strand primer P3-t (SEQ ID NO: 6) located on the short arm
CR was performed. The PCR product was electrophoresed on an agarose gel and then Hybond N + (Amersham Pharmacia Biotec
Alkaline transfer to h). The Southern blot thus prepared was analyzed by Southern hybridization using the 0.4 kbp PvuII fragment in the short arm region as probe B. For clones in which a band at 1.2 kbp was detected, genomic DNA was extracted from cells that had been subsequently scaled up and cultured, and treated with EcoRI restriction enzyme to obtain LLPL cDNA 3 '.
-0.5 kbp of the region outside the short arm existing in the untranslated region
Genomic Southern analysis was carried out using the HindIII / PstI fragment of E. coli as probe C. 6.8 kbp in the LLPL gene knockout cell line (Disrupted allele (Disrupted
allele)) and 7.5 kbp (Wild-type a
A band could be detected at the position (llele)). On the other hand, only the 7.5 kbp (Wild-type allele) band could be detected in the control wild strain. 7.5 kbp and Di from wild allele
The one in which two 6.8 kbp bands derived from srupted allele were detected was finally selected as a homologous recombinant of interest. Two experiments were conducted to obtain homologous recombinants.
It was confirmed that 5 clones out of a total of 430 clones showing resistance to both 418 and FIAU were homologous recombinants in which a mutation was introduced (FIG. 3).

Example 2 Production of chimeric mouse using homologous recombinant ES cell LX-2-163 strain 1. Acquisition of blastocyst A blastocyst collecting animal was produced as follows. First, we purchased 8-week-old female C57BL / 6 mice (Crj) at room temperature of 25 ± 1
The animals were acclimated for 1 week at an SPF animal breeding facility controlled at 12 ° C., a humidity of 50 ± 10%, and 7 hours to 7 hours of daylight conditions. On the first day of the experiment, they were allowed to cohabit with male mice of the same strain aged 10 weeks or older and spontaneously mated. On the next day, vaginal plugs of female mice cohabiting with each other were confirmed, and female mice with confirmed vaginal plugs were bred for 3 days until the microinjection experiment. On the day of the microinjection experiment, the mice were sacrificed by cervical dislocation, the abdomen was opened, and the ovaries, fallopian tubes, and uterus were taken out. The upper part of the uterine horn was removed, and the uterus was divided into left and right parts. Then, insert 25G injection needles from the uterine horn side and the vaginal side, and perfuse with egg collection medium (DMEM containing 10% FCS, 100 U penicillin and 100 U streptomycin), and perfuse the perfusate of 3.5 per mouse. cm collected in a petri dish, 37 ℃, 7% CO ₂
After leaving it in the incubator under the conditions for 30 minutes or more, only the blastocyst was recovered. 2. Preparation of homologous recombinant ES cells The homologous recombinant ES cells that had been cryopreserved at a cell density of 5 × 10 ⁵ or 1 × 10 ⁶ were thawed on the second day of the experiment and immediately S
Use TO cells in feeder medium (10% FCS, 200 mM L-glutamin
e, 10 mM NEAA, 100 U penicillin and 100 U streptomycin-containing DMEM) 6 cm
Seed on diameter feeder dish. 37 ° C, 7% CO ₂
ES medium (16% FCS, 2%) until the day of microinjection experiment in an incubator controlled by conditions.
00mM L-glutamine, 10mM NEAA, 100U Penicillin, 100
U streptomycin, 1M HEPES, 1000U ESGRO and 0.
The cells were cultured in DMEM containing 1 mM β-mercaptoethanol for 3 days. The homologous recombinant ES cells that had proliferated were washed twice with a PBS solution after removing the medium. The washed cells were treated with a 0.025% trypsin solution for 5 minutes, added with the above ES medium, and then centrifuged at 1200 rpm for 2 minutes at 4 ° C. Next, the supernatant was removed, 2-3 ml of ES medium was added, the cells were well loosened, and the same medium was further added to adjust the volume to 10 ml. The above-mentioned cell suspension was seeded on a culture dish with a diameter of 10 cm, and then cultured for 30 minutes in an incubator controlled at 37 ° C. and 7% CO ₂ conditions to adhere feeder cells to the culture dish. Further, only ES cells contained in the supernatant were collected in a 6 cm-diameter Petri dish and stored at 4 ° C until the start of the microinjection experiment. 3. Microinjection experiment of homologous recombinant ES cells As an injection chamber, a medium for microinjection (1
6% FCS, 200 mM L-glutamine, 10 mM NEAA, 100 U penicillin, 100 U streptomycin, 20 mM HEPES and 0.1 m
One drop of (DMEM containing M β-mercaptoethanol) was added dropwise, 5 to 10 blastocysts were transferred to the center of the drop, and the homologous recombinant ES cells were transferred to the periphery of the drop.
Add liquid paraffin to prevent the drop from drying, and
Refrigerated at ℃ for 20-30 minutes. The microinjection experiment was performed by the method of Hogan et al. (Hogan, B., Costanitini, F., and La.
cy, E. (Eds). 1986. Manipulation the mouse embryo;
A laboratory manual. New York: Cold Spring Harbor
Laboratory Press.). Homologous recombinant E
For microinjection of S cells into the blastocyst, 10 to 15 ES cells in the drop are collected with a microinjection pipette, and then the above microinjection pipette is used from the position opposite to the inner cell mass of the blastocyst. It was carried out by inserting into the cavity and injecting. Blastocysts that have completed microinjection are D supplemented with 10% FCS.
The cells were transferred to MEM medium and cultured in an incubator controlled at 37 ° C. and 7% CO ₂ conditions until the blastocysts returned to their original shape. Normal blastocyst is ICR prepared by the following method
(JcL) 8 to 10 mice each were transplanted to the left and right uterine horns of pseudopregnant female mice, and the room temperature was 25 ± 1 ° C, the humidity was 50 ± 10%, and the hours were 7 to 19:00 in a breeding room controlled by daylight conditions The transplanted mice were bred. Pseudopregnant female mice were produced as follows. Purchased 8-week-old ICR (JcL) female, room temperature 25 ± 1 ℃, humidity 50 ± 10
%, And acclimation for 1 week at an SPF animal breeding facility controlled under 12-hour sunshine conditions at 7 to 19 o'clock. On the second day of the experiment, the mice were allowed to cohabit with ICR (JcL) strain vas deferens male mice of 10 weeks or older prepared in advance and spontaneous mating was performed. On the next day, vaginal plugs of female mice cohabiting with each other were confirmed, and the mice were bred for 2 days until the day of the microinjection experiment and used for the experiment. As a result, 49 pups were obtained in the microinjection experiment of strain LX-2-163. 27 of them were chimeric mice with agouti-colored hair. Inter-individual variation was observed in the contribution rate (chimera rate) of ES cells to the body hair color of the chimeric mouse. In the same experiment, 10 male chimeric mice having a high chimera rate of 75% or more were obtained.

Example 3 Production of LLPL-deficient mice Only male chimera mice showing a high chimera rate were aged 10 weeks or older.
Mating was performed with C57BL / 6 strain (CrJ) females. First, the determination of the transfer of ES cells to the germ line was performed by a method for confirming the color of offspring. LX-2-163 cell line contribution is almost 10
All 75 offspring obtained from the crossing with 0% chimeric mice showed Agouti color, and all the offspring were considered to be derived from ES cells. Of 120 pups obtained from a cross with a male chimera mouse having a high chimera rate of 75% or more, 80 of them showed an agouti color derived from ES cells.
Next, the gene deficiency was determined by the following PCR method and Southern analysis in mice exhibiting agouti body color. DN from the tail of an ES cell-derived offspring mouse according to a standard method
A was extracted, and using this genomic DNA as a template, the sense strand primer SI-75 [5'-GAT TG on the neomycin resistance gene
G GAA GAC AAT AGC AGG CAT GC-3 '; 26-mers (SEQ ID NO: 5)] and sense strand primer P4- on the LLPL gene
s [5'-CGC TCA TCT GAT CAT CGT AAT GAT CG-3 '; 26-mer
s (SEQ ID NO: 7)] and the antisense strand primer P3-t [5'-TGG TGC AAG TAT TAT CA located on the short arm
C CGC CCA TT-3 '; 26-mers (SEQ ID NO: 6)] was used for PCR, and analyzed by agarose gel. Using this primer, amplification of a 0.4 kbp band was observed from Wild-type allele in which no mutation was introduced, and Disrup in which a mutation was introduced into the LLPL gene
Amplification of the 0.3 kbp band is seen from tedallele. Of 28 cases (12 males and 16 females) from chimeric mice (F0) and C57BL / 6J, 16 bands (6 males and 10 females) showed amplification of two bands of 0.3 kbp and 0.4 kbp. It was observed and considered to be a heterozygous mouse in which a mutation (deficiency) was introduced in the LLPL gene. These mouse DN
After A was treated with EcoRI, it was analyzed by genomic Southern hybridization using probe C in the same manner as in [Example 1]. LLPL obtained in [Example 1]
7.5 kbp and the same as gene-deficient ES cell line LX-2-163
A band was detected at the position of 6.8 kbp, confirming that the mouse was a heterozygous mouse (F1).

Example 4 Properties of LLPL-deficient mouse By mating the male and female of the LLPL hetero-deficient mouse (F1) obtained in Example 3, an LLPL homo-deficient mouse (F2) can be obtained with a probability almost according to Mendelian rule. It was FIG. 4 shows the result of analysis by the Southern hybridization method similar to that of [Example 3]. In LLPL homo-deficient mice
Only the 6.8 kbp band was detected (Fig. 4). further,
Male F2 mice were injected intraperitoneally with 1.5 ml of thioglycollate. After 4 days, the cervical spine was dislocated and the peritoneal macrophages were suspended in 10 ml of D-PBS (-) and collected.
From the obtained peritoneal macrophages, poly (A) ⁺ RNA was purified, and Northern blot analysis was performed using 0.5 kbp DNA of the N-terminal region of LLPL ORF as a probe.
It was shown to. Original L in wild type mice (LLPL ^{+ / +} )
A 3 kbp band derived from the LPL gene was observed, and a 3 kbp band in the hetero mouse (LLPL ^+/- ) and a 2 kbp band corresponding to the active site-deficient LLPL gene were observed.
In homozygous mice (LLPL ^-/- ), LL lacking active site
Only the 2 kbp band corresponding to the PL gene was detected. That is, it was confirmed at the mRNA level that expression of a short mRNA lacking the active site of the LLPL gene was observed in the LLPL homo-deficient mouse (FIG. 5). Peritoneal macrophages obtained by the same method as above were treated with DMEM (10% FBS).
After culturing overnight in 10 cm and adhering to a 10 cm dish, DM
The medium was replaced with 5 ml of EM serum-free medium and cultured for 5 days. The culture supernatant was collected and 0.22 μm filter sterilized, and the sample was subjected to immunoprecipitation of mLLPL protein. After adding 10 μl of 10% SDS to 900 μl of the culture supernatant sample, the protein was denatured by heating at 100 ° C. for 2 minutes.
20mM Tris-HCl (pH7.4), 150mM NaCl, 10% Trito
Polyclonal antibody PCL02 of hLLPL protein that crosses 100 μl of n X-100, 0.1% NaN ₃ solution and mLLPL protein
Alternatively, add 10 μg of rabbit IgG and add 1.5 μg at 4 ° C overnight.
Spin in ml Eppendorf tube. More anti-r
50 μl of abbit IgG agarose (1: 1 slurry) (Sigma) was added and spun in a 1.5 ml Eppendorf tube overnight at 4 ° C. 20 mM Tris-HC on the agarose gel after immunoprecipitation
l (pH7.4), 150mM NaCl, 1% Triton X-100, 1mg / ml BS
A, washed 3 times with 0.1% NaN ₃ solution, then TBS, 50m
It was washed once with MTris-HCl (pH 7.4). After washing 2 x Laem
20 μl of the sample that was obtained by elution of protein in 40 μl of mli Sample Buffer (Bio-Rad) and denaturation by heating at 100 ° C for 5 minutes
Was used for SDS-PAGE. After performing electrophoresis according to a conventional method, Western blotting was performed on a Hybond-P (Amersham Pharmacia Biotech) membrane. Biotinylated PCL02 antibody as the primary antibody and streptavedine AP-conjugate as the secondary antibody.
(Boehringer Manheim)) was used for Western analysis. The mouse LLPL protein shows a broad band around about 50 kDa, but upon decomposition, it was detected as multiple bands in the region of less than 50 kDa. Wild type LLPL protein
(About 50 kDa) and the mutant LLPL protein lacking the active site that was expressed in mRNA (if produced as a protein, it is expected to be about 15 kDa), the protein Western analysis was carried out using Multi-Gel 12.5 and Multi-Gel 15/25 (Daiichi Pure Chemicals) having different image ranges. Figure 9 shows Western blot analysis using Multigel 12.5. The LLPL protein (50-60 kDa
(Around the band) was detected, but no band was detected around 50 kDa in homo, and even in the case of using Multigel 15/25, a band that appears to be derived from a short mutant protein near 15 kDa in both homo and hetero. Was not detected (Fig. 6). Therefore, it was confirmed that at least an active LLPL protein was not produced. Although the defective mouse produced this time synthesizes incomplete mRNA, it is considered that the protein produced therefrom is unstable and does not exist.

No abnormalities were observed in the LLPL homo-deficient mice, and no apparent difference in postnatal weight increase was observed among the homo, hetero, and wild type groups. LLPL
Third-generation male and female LLPL homo-deficient mice derived from the gene-deficient ES cell line LX-2-163 and wild-type mice were fed with a normal diet or a high-fat high-cholesterol diet for 16 weeks from the age of 6 weeks. As a result, LLP fed a normal diet
The plasma cholesterol of L-hom-deficient mice was not significantly different from that of wild-type mice, but showed a low tendency. On the other hand, when a high-fat high-cholesterol diet is given, total cholesterol and LDL + VLDL cholesterol in plasma, triglyceride in the liver, phospholipids,
Free cholesterol, total cholesterol, and liver weight were increased in both LLPL homozygous and wild type mice. However, in all the items, the LLPL homo-deficient mice tended to have lower levels than the wild-type mice. This phenomenon was more apparent in males than in females. Moreover, histopathological examination showed that by feeding a high-fat and high-cholesterol diet for 16 weeks, hepatocytes increased vacuoles due to excessive fat accumulation and disordered cord structures, increased single cell necrosis, and large and small nuclear cells. Findings of fatty liver such as nuclear infiltration were found in LLPL homo-deficient mice and wild-type mice. In the case of males, the degree of fatty liver is LLP.
L-homodeficient mice were milder than wild-type mice (FIG. 7), consistent with findings in blood lipids and liver lipids.
Of the lipid content in the liver of male homozygous individuals, triglyceride, phospholipid, and free cholesterol showed significantly low values when compared with the wild type group (FIG. 8).

Example 5 Generation of LLPL / ApoE double-deficient mouse F1 heterozygotes were obtained by mating LLPL-deficient male homozygous mice (LLPL ^{− / −} ) with ApoE-deficient female homozygous mice (ApoE ^{− / −} ). Body-deficient mice (LLPL ^+/- / ApoE ^+/- ) were created. Next, this F1 heterozygous deficient mouse was crossed,
In the PCR determination of the obtained F2 mice, wild type mice (LLPL ^{+ / +} / ApoE ^{+ / +} ) and LLPL deficient mice (LLPL ^-/- / ApoE
^{+ / +} ), ApoE deficient mice (LLPL ^{+ / +} / ApoE ^-/- ), and LLPL
/ ApoE double deficient mice (LLPL ^-/- / ApoE ^-/- ) were selected. After that, production was carried out by crossing each of the above individuals.

Example 6 Evaluation of Atherosclerotic Lesions in Aorta of LLPL / ApoE Double Deficient Male Mice Wild type mice (LLPL ^{+ / +} / ApoE ^{+ / +} ) and LLPL deficient mice (L
LPL ^-/- / ApoE ^{+ / +} ), ApoE deficient mouse (LLPL ^{+ / +} / Apo
E ^-/- ), LLPL / ApoE double deficient mouse (LLPL ^-/- / ApoE ^{-/ -} )
4 male mice were bred with a normal diet (CE-2 solid feed: CLEA Japan, Inc.). At the age of 18 weeks, the animals were sacrificed by decapitation, and the heart and aorta were collected from each individual. After sacrifice, liver, small intestine, lungs, visceral tissues such as fat were roughly removed, and fat and connective tissue around the aorta from the arch to the thigh were removed under a stereoscopic microscope. The aorta was cut open from the abdomen toward the arch and the thigh, and the base of the heart and the branched part of the thigh were excised to take out the aorta. The collected aorta was carefully removed of the adipose tissue and connective tissue attached to the outside in saline, and the coagulated blood was carefully removed, and the lumen side of the aorta was attached to the black rubber plate with insect pins to prepare a specimen. .
Regarding the prepared aortic specimen, Sudan IV
Lipid staining was performed. After gently rinsing the specimen with the rubber plate with 70% ethanol, 0.5% Sudan IV (aceton: EtO
(H: H ₂ O = 1: 1: 1) Dipped in a solution and stained for 30 minutes. The sample was destained with 70% ethanol, washed with water, fixed in 4% formalin / PBS, and photographed with a digital camera. The total area and the lesion area in the aortic specimen were quantified using the image analysis application MacScope. FIG. 10 shows the lesion area ratio in the aortic specimen of each group. Wild type mouse (LLPL ^{+ / +} / ApoE
^{+ / +} ) And LLPL deficient mice (LLPL ^-/- / ApoE ^{+ / +} ) showed no formation of atherosclerotic lesions. The area ratio of arteriosclerotic lesions in ApoE-deficient mice (LLPL ^{+ / +} / ApoE ^{-/ -} : n = 6) is (1.8 ± 1.0)%, whereas LLPL / ApoE double-deficient mice (LLPL- ^{/ -} / ApoE ^-/- : n = 5) was (4.2 ± 2.1)%, which was a 2.3-fold significant increase (P <0.05; by Dunnet
t test).

Example 7 Changes in Plasma Total Cholesterol Levels in LLPL / ApoE Double-Deficient Male Mice For the mice used in the experiment of Example 6, 6, 8, 10, 1
Blood was collected from the orbital venous clot using a glass capillary containing sodium heparin at 2, 14, 16 and 18 weeks of age. Figure 1 shows ApoE-deficient mice (LLPL ^{+ / +} / ApoE ^-/- ), LLPL /
Data of both groups of ApoE double deficient mice (LLPL ^-/- / ApoE ^-/- ) are shown. From changes in plasma cholesterol levels over 6-18 weeks of age in both groups, LLPL / ApoE double deficient mice (LLPL ^-/- / A
The plasma total cholesterol level of poE ^-/- ) tended to be slightly higher.

Example 8 Evaluation of Atherosclerotic Lesions in Aorta of LLPL / ApoE Double Deficient Female Mice Wild type mice (LLPL ^{+ / +} / ApoE ^{+ / +} ) and LLPL deficient mice (L
LPL ^-/- / ApoE ^{+ / +} ), ApoE deficient mouse (LLPL ^{+ / +} / Apo
E ^-/- ), LLPL / ApoE double deficient mouse (LLPL ^-/- / ApoE ^{-/ -} )
4 groups of female mice were bred on a normal diet (CE-2 solid feed: CLEA Japan, Inc.) up to 38 weeks of age. At 38 weeks of age, the animals were sacrificed by cervical dislocation, and aortas were collected from each individual. The method of collecting mouse aorta and staining the lipid thereof was carried out according to Example 5. FIG. 12 shows the lesion area ratio in the aortic specimen of each group. Wild-type mouse (LLPL ^{+ / +} / ApoE ^{+ / +} ) and LLPL
In the deficient mice (LLPL ^-/- / ApoE ^{+ / +} ), atherosclerotic lesions were not formed at all, as in 18-week-old males. ApoE
The area ratio of arteriosclerotic lesions in deficient mice (LLPL ^{+ / +} / ApoE ^-/- : n = 4) is (18.3 ± 2.1)%, whereas LLPL / A
In poE double-deficient mice (LLPL ^-/- / ApoE ^-/- : n = 4), (49.3
± 3.2)%, which was a 2.7-fold significant increase (P <0.0
1; by Dunnett test).

Example 9 Changes in Plasma Cholesterol Levels of LLPL / ApoE Double-Deficient Female Mice 6, 8, 10, 1 of the mice used in the experiment of Example 8
Blood was collected from the orbital venous plexus at 4, 18 and 28 weeks of age using a glass capillary containing sodium heparin.
Wild type mouse (LLPL ^{+ / +} / ApoE ^{+ / +} ), LLPL deficient mouse (L
LPL ^-/- / ApoE ^{+ / +} ) between both groups and ApoE-deficient mice (LL
PL ^{+ / +} / ApoE ^-/- ), LLPL / ApoE double deficient mouse (LLPL ^-/- /
ApoE ^-/- ) groups showed almost the same changes in plasma cholesterol levels up to 28 weeks of age, with no significant difference. ApoE deficient mouse (LLPL ^{+ / +} / Apo
E ^-/- ), LLPL / ApoE double-deficient mouse (LLPL ^-/- / ApoE ^-/- )
The data of both groups are shown in FIG.

[0034]

INDUSTRIAL APPLICABILITY The non-human animal ES cell in which the LLPL gene is inactivated according to the present invention is very useful in producing a non-human animal deficient in LLPL gene expression. L of the present invention
Since non-human animals deficient in LPL expression lack various biological activities that can be induced by LLPL, they can serve as models of diseases caused by inactivation of biological activity of LLPL. It is useful for screening prophylactic and / or therapeutic agents, investigating the cause of LLPL-related diseases, and examining therapeutic methods. In addition, L of the present invention
A dual-function deficient model animal produced by mating a non-human LPL expression-deficient animal with an arteriosclerosis model animal has a relationship with a disease associated with arteriosclerosis-related gene ApoE, prevention of various diseases caused by LLPL deficiency or ApoE deficiency, and / or Alternatively, it is useful for screening therapeutic agents, investigating the causes of LLPL and ApoE related diseases, and examining therapeutic methods thereof. The LLPL gene-introduced non-human animal of the present invention has high LLPL disease, LL
It is possible to elucidate the pathological mechanism of LLPL-related diseases such as PL dysfunction and to study treatment methods for these diseases. In addition, it can be used for screening therapeutic agents for the above LLPL-related diseases. The screening method of the present invention enables efficient screening of preventive and / or therapeutic agents for various diseases caused by LLPL deficiency.

[0035]

[Sequence list] <110> Takeda Chemical Industries, Ltd. <120> knockout animals <130> P02-0063 <150> JP2001-152520 <151> 2001-05-22 <150> JP2001-311971 <151> 2001-10-09 <160> 7 <210> 1 <211> 412 <212> PRT <213> Mouse <400> 1 Met Asp Arg His Leu Cys Thr Cys Arg Glu Thr Gln Leu Arg Ser Gly   1 5 10 15 Leu Leu Leu Pro Leu Phe Leu Leu Met Met Leu Ala Asp Leu Thr Leu              20 25 30 Pro Ala Gln Arg His Pro Pro Val Val Leu Val Pro Gly Asp Leu Gly          35 40 45 Asn Gln Leu Glu Ala Lys Leu Asp Lys Pro Lys Val Val His Tyr Leu      50 55 60 Cys Ser Lys Lys Thr Asp Ser Tyr Phe Thr Leu Trp Leu Asn Leu Glu  65 70 75 80 Leu Leu Leu Pro Val Ile Ile Asp Cys Trp Ile Asp Asn Ile Arg Leu                  85 90 95 Val Tyr Asn Arg Thr Ser Arg Ala Thr Gln Phe Pro Asp Gly Val Asp             100 105 110 Val Arg Val Pro Gly Phe Gly Glu Thr Phe Ser Met Glu Phe Leu Asp         115 120 125 Pro Ser Lys Arg Asn Val Gly Ser Tyr Phe Tyr Thr Met Val Glu Ser     130 135 140 Leu Val Gly Trp Gly Tyr Thr Arg Gly Glu Asp Val Arg Gly Ala Pro 145 150 155 160 Tyr Asp Trp Arg Arg Ala Pro Asn Glu Asn Gly Pro Tyr Phe Leu Ala                 165 170 175 Leu Arg Glu Met Ile Glu Glu Met Tyr Gln Met Tyr Gly Gly Pro Val             180 185 190 Val Leu Val Ala His Ser Met Gly Asn Val Tyr Met Leu Tyr Phe Leu         195 200 205 Gln Arg Gln Pro Gln Val Trp Lys Asp Lys Tyr Ile His Ala Phe Val     210 215 220 Ser Leu Gly Ala Pro Trp Gly Gly Val Ala Lys Thr Leu Arg Val Leu 225 230 235 240 Ala Ser Gly Asp Asn Asn Arg Ile Pro Val Ile Gly Pro Leu Lys Ile                 245 250 255 Arg Glu Gln Gln Arg Ser Ala Val Ser Thr Ser Trp Leu Leu Pro Tyr             260 265 270 Asn His Thr Trp Ser His Glu Lys Val Phe Val Tyr Thr Pro Thr Thr         275 280 285 Asn Tyr Thr Leu Arg Asp Tyr His Arg Phe Phe Arg Asp Ile Gly Phe     290 295 300 Glu Asp Gly Trp Phe Met Arg Gln Asp Thr Glu Gly Leu Val Glu Ala 305 310 315 320 Met Thr Pro Pro Gly Val Glu Leu His Cys Leu Tyr Gly Thr Gly Val                 325 330 335 Pro Thr Pro Asn Ser Phe Tyr Tyr Glu Ser Phe Pro Asp Arg Asp Pro             340 345 350 Lys Ile Cys Phe Gly Asp Gly Asp Gly Thr Val Asn Leu Glu Ser Val         355 360 365 Leu Gln Cys Gln Ala Trp Gln Ser Arg Gln Glu His Arg Val Ser Leu     370 375 380 Gln Glu Leu Pro Gly Ser Glu His Ile Glu Met Leu Ala Asn Ala Thr 385 390 395 400 Thr Leu Ala Tyr Leu Lys Arg Val Leu Leu Glu Pro                 405 410 <210> 2 <211> 1236 <212> DNA <213> Mouse <400> 2 atggatcgcc atctctgcac ctgtcgcgag acccagctcc ggagtggcct cctgttacct 60 ctgtttctac taatgatgct ggcagacctg acgctcccgg cccaacgtca ccccccggtg 120 gtgctggtgc ctggtgattt gggtaaccag ttggaagcaa agctggataa gccaaaggtt 180 gtacactacc tttgctccaa gaagacggac agctacttca cactctggct gaatctggaa 240 ctgcttctgc ctgttatcat tgactgctgg attgacaata tcaggctggt ttacaacaga 300 acatctcggg ccacccagtt tcccgatggt gtggacgtgc gtgtccctgg ctttggggaa 360 acattttcta tggaattcct agaccccagc aagaggaatg tgggttccta tttctacact 420 atggtggaga gccttgtggg ctggggctac acacggggtg aagacgttcg aggtgctccc 480 tatgattggc ggcgagcccc aaatgaaaac gggccctact tcttggccct gcgagagatg 540 atcgaggaga tgtaccagat gtatgggggc cccgtggtgc tggtcgccca cagcatgggc 600 aacgtgtaca tgctctactt tctgcagcgg cagccacaag tctggaagga caaatatatc 660 catgccttcg tctcactggg ggcgccctgg gggggcgtgg ccaagacgct gcgtgtcctg 720 gcctcaggag acaacaatcg cattcccgtc attgggccac tgaagatccg ggaacagcag 780 cgatctgccg tctctaccag ctggctactg ccatacaacc acacttggtc acatgaaaag 840 gtatttgtat acacacccac gactaactac acgctccggg actatcaccg gttcttccgg 900 gacatcggtt tcgaagatgg ctggttcatg cggcaggaca cagaagggct ggttgaagcc 960 atgacgccac ccggggtgga gctgcactgc ttgtatggca ctggtgttcc cacgccaaac 1020 tctttctact acgagagctt tcctgatcgg gaccccaaaa tctgcttcgg cgatggtgac 1080 ggcacggtga acctggagag cgtcctgcag tgccaagcct ggcagagccg ccaagagcac 1140 agagtatcat tgcaggagct gccgggaagc gagcacattg agatgctagc caatgccacc 1200 accttggctt atctgaaacg tgtgcttctg gaacct 1236 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer P94-1 <400> 3 ggtaaccagt tggaagcaaa g 21 <210> 4 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer P101-4 <400> 4 ccccgggtgg cgtcat 16 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer SI-75 <400> 5 gattgggaag acaatagcag gcatgc 26 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer P3-t <400> 6 tggtgcaagt attatcaccg cccatt 26 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer P4-s <400> 7 cgctcatctg atcatcgtaa tgatcg 26

[0036]

[Brief description of drawings]

FIG. 1 shows the exon and intron structures of the LLPL gene on the mouse genome. In the figure, the exon portion is shown by a box and the intron portion is shown by a solid line. In addition, 5 DNAs obtained by restriction enzyme treatment and subcloned
The fragment is indicated by a thick solid line.

FIG. 2 shows a construction diagram of the targeting vector obtained in Reference Example 3. In the figure, Neo ^r indicates the neomycin resistance gene, and HSV-TK indicates the herpes simplex virus-thymidine kinase gene.

FIG. 3 shows LLPL knockout E obtained in Example 1.
The result of Southern hybridization of S cell line is shown. Transgenic ES cell line: LX-1-5 (lane 1); LX-1-8 (lane 2); LX-2-3 (lane 3); LX-2
-80 (lane 4); LX-2-163 (lane 5); and wild type ES cell line (lanes 6, 7); STO cells (lane 8).

FIG. 4 shows LLPL-deficient mice obtained in Example 4 (F
The result of Southern hybridization of 2) is shown.

FIG. 5 shows the results of Northern blotting analysis of LLPL expression in peritoneal macrophages of LLPL-deficient mice obtained in Example 4. In the figure, + / + is wild type,
+/− indicates a hetero-deficient mouse and − / − indicates a homo-deficient mouse.

FIG. 6 shows the result of analysis of LLPL produced in the peritoneal macrophage culture supernatant of the LLPL-deficient mouse obtained in Example 4 by Western blotting via immunoprecipitation. In the figure, + / + indicates wild type, +/− indicates hetero-deficient mouse, and − / − indicates homo-deficient mouse.

FIG. 7 shows histopathological examination of the livers of the wild type group fed with a high-cholesterol high-fat diet for 16 weeks and the homozygous group obtained in Example 4. The above figure is 100 times for hematoxylin and eosin (H & E) stained specimens. The figure below is 100 times for a fat-stained (Oil Red O) specimen. In the wild-type group, intrahepatic cytoplasmic vacuolation of the lobules, disordered cord-like structures, large and small distribution of hepatocyte nuclei due to hepatocyte regeneration, single cell necrosis of hepatocytes and mononuclear cell infiltration and diffuse accumulation of neutral fat. Can be seen. In the homozygous population, centrilobular intrahepatic cytoplasmic vacuolization and hepatocellular nuclei size variation were observed, and centrilobular triglyceride accumulation and fat accumulation were reduced compared to the wild type.

FIG. 8 shows the lipid content per wet weight of each group.

FIG. 9 shows the results of analysis of LLPL produced in the peritoneal macrophage culture supernatant of LLPL-deficient mice obtained in Example 4 by Western blotting (using Multigel 12.5) via immunoprecipitation. In the figure, + / + indicates wild type, +/− indicates hetero-deficient mouse, and − / − indicates homo-deficient mouse.

FIG. 10 is a comparison of the area ratio of arteriosclerotic lesions stained with Sudan IV in the aorta of LLPL / ApoE double deficient mice, ApoE deficient mice, LLPL deficient mice, and wild-type mice (18-week-old male).

FIG. 11 shows changes in plasma total cholesterol levels over time in LLPL / ApoE double deficient mice and ApoE deficient mice (18-week-old males).

FIG. 12: LLPL / ApoE double deficient mouse, ApoE deficient mouse, LLPL deficient mouse, wild type mouse (38-week-old female)
Of Area Ratio of Atherosclerotic Lesions Stained with Sudan IV in Human Aorta

FIG. 13: Time-course changes in plasma total cholesterol levels in LLPL / ApoE double-deficient mice and ApoE-deficient mice (38-week-old females)

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 43/00 105 C12Q 1/02 C12N 5/10 G01N 33/15 Z C12Q 1/02 33/50 Z G01N 33/15 C12N 15/00 ZNAA 33/50 5/00 B (72) Inventor Mitsugu Nakata 1-22-5 Kiyomidai, Kawachinagano City, Osaka Prefecture (72) Inventor Yoshitaka Yasuhara 18 Kuwatacho, Ibaraki City, Osaka Prefecture No. 6 (72) Inventor Kao Taniyama 1-7-7 Kasuga, Tsukuba, Ibaraki 9 Takeda Kasuga Heights 501F Term (reference) 2G045 AA40 4B024 AA01 AA11 CA04 DA20 EA04 GA14 HA20 4B063 QA01 QA05 QA18 QA19 QQ08 QQ76 QR72 QR72 QX01 4B065 AA90X AA91Y AB01 BA03 CA44 CA46 4C084 AA17 NA14 ZA451 ZB212 ZC331

Claims (26)

[Claims] 1. A mammalian embryonic stem cell in which a lecithin: cholesterol acyltransferase-like lysophospholipase gene is inactivated. 2. The embryonic stem cell according to claim 1, which is drug resistant. 3. The embryonic stem cell according to claim 2, wherein the drug is neomycin. 4. The embryonic stem cell according to claim 1, wherein the lecithin: cholesterol acyltransferase-like lysophospholipase gene is inactivated by inserting a reporter gene. 5. The embryonic stem cell according to claim 4, wherein the reporter gene is the lacZ gene. 6. The embryonic stem cell according to claim 1, wherein the mammal is a rodent. 7. The embryonic stem cell according to claim 6, wherein the rodent is a mouse. 8. A non-human mammal deficient in expression of a lecithin: cholesterol acyltransferase-like lysophospholipase gene, a tissue thereof, or a cell derived therefrom. 9. The animal according to claim 8, the tissue thereof or cells derived therefrom, wherein the non-human mammal is a rodent. 10. A preventive and / or therapeutic agent for a disease caused by a deficiency of a lecithin: cholesterol acyltransferase-like lysophospholipase gene, which comprises using the animal according to claim 8 or a tissue thereof or cells derived from them. Screening method. 11. Prevention of a disease caused by a deficiency of lecithin: cholesterol acyltransferase-like lysophospholipase gene, which comprises administering a test compound to the animal according to claim 8 or a tissue thereof or cells derived therefrom. / Or a method for screening therapeutic agents. 12. The screening method according to claim 10, wherein the disease is arteriosclerosis. 13. A preventive and / or therapeutic agent for a disease caused by a deficiency in a lecithin: cholesterol acyltransferase-like lysophospholipase gene, which is obtained by the screening method according to claim 10. 14. The preventive and / or therapeutic drug according to claim 13, wherein the disease is arteriosclerosis. 15. A disease model animal or tissue thereof produced by mating of the animal of claim 8 with another disease model animal, or a cell derived therefrom. 16. A disease state model animal or its tissue, or a cell derived therefrom, which is caused by drug induction or stress load on the animal of claim 8. 17. The pathological model animal according to claim 15, the tissue thereof, or cells derived therefrom, wherein the other pathological model animal is a non-human mammal deficient in apolipoprotein gene expression. 18. The pathological model animal according to claim 17, its tissue or its origin, wherein the non-human mammal is a non-human mammal deficient in lecithin: cholesterol acyltransferase-like lysophospholipase gene and apolipoprotein gene. cell. 19. The animal model for pathological conditions according to claim 17, the tissue thereof or cells derived therefrom, wherein the apolipoprotein is apolipoprotein E. 20. A disease state model animal or its tissue or a cell derived therefrom, which is caused by drug induction or stress load on the animal according to claim 15. 21. Due to a deficiency of a lecithin: cholesterol acyltransferase-like lysophospholipase gene, characterized by using the animal or the tissue thereof or the cell derived therefrom according to any one of claims 15 to 17 or 20. A method for screening a drug for preventing and / or treating a disease. 22. A lecithin: cholesterol acyltransferase-like lysophospholipase gene, which comprises administering a test compound to the animal or the tissue thereof or the cells derived therefrom according to any of claims 15 to 17 or 20. A method for screening a preventive and / or therapeutic drug for a disease caused by a defect. 23. A preventive and / or therapeutic agent for a disease caused by a deficiency of a lecithin: cholesterol acyltransferase-like lysophospholipase gene obtained by the screening method according to claim 22 or 23. 24. A preventive and / or therapeutic agent for a disease caused by a deficiency of a lecithin: cholesterol acyltransferase-like lysophospholipase gene, which comprises using the animal according to claim 17, its tissue, or cells derived therefrom. Screening method. 25. Prevention of a disease caused by a deficiency of lecithin: cholesterol acyltransferase-like lysophospholipase gene, which comprises administering a test compound to the animal according to claim 17, its tissue or cells derived therefrom. / Or a method for screening therapeutic agents. 26. A preventive and / or therapeutic agent for a disease caused by a deficiency of a lecithin: cholesterol acyltransferase-like lysophospholipase gene obtained by the screening method according to claim 24 or 25.