JP2006141329A - POLtheta-KNOCKOUT ANIMAL, COMPOSITION FOR REGULATING IMMUNE FUNCTION AND METHOD FOR SCREENING THE SAME - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a useful means for the functional analysis of Polθ gene, a new medicine or reagent and a useful means for developing them. <P>SOLUTION: This non-human animal containing the functional deletion of the Polθ gene and a method for producing the same; animal cells containing the functional deletion of the Polθ gene and a method for producing the same; a targeting vector for inducing a homologous recombinant of the Polθ gene and a method for producing the same; a composition capable of regulating an immune function; a method for screening a substance capable of regulating the immune function; a method for identifying the polymorphism of the Polθ gene bringing the change of the regulating activity of the immune function; a method for judging the onset or the risk of onset of immune diseases in the animals; a diagnostic agent for the onset or the risk of onset of the immune diseases, etc., are provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention provides a novel animal, a novel animal cell, a novel targeting vector, and a production method thereof, and a novel composition, a novel screening method, a novel identification method, a novel determination method, a novel diagnostic agent, and the like.

  In recent years, elucidation and analysis of genome sequences of various organisms have been advanced at a global level. For example, in humans and mice, analysis of the entire genome sequence has been completed. Genomic information is considered to have very important significance in drug discovery, but simply determining the genome sequence does not reveal the function of the gene and find the target protein for drug discovery.

  A gene knockout technique is known as one of the techniques for elucidating the function of a gene. According to the gene knockout technique, important information that cannot be obtained only by genome sequence analysis can be obtained, and a novel drug discovery target protein can be discovered.

  The Polθ (DNA polymerase θ) gene is a gene encoding a protein containing a helicase domain at its N-terminal part and a Pol domain at its C-terminal part (see, for example, GenBank accession number: AY074936. (Also referred to as polq, it is referred to as the Polθ gene for convenience in this specification). Drosophila Polθ mutant has been reported to be highly sensitive to bifunctional DNA crosslinkers such as cisplatin, but not to monofunctional crosslinkers such as methylmethanesulfonate, which indicates that the Polθ gene is It is considered necessary for repair of DNA interstrand crosslinks. In addition, Drosophila Polθ mutants have also been reported to dramatically increase spontaneous chromosomal abnormalities. These findings indicate that the Polθ gene plays a role in maintaining genome stability as well as DNA repair.

  Human Polθ exhibits 42% identity (62% similarity) in the helicase domain and 27% identity (41% similarity) in the Pol domain relative to Drosophila Polθ. The function of human Polθ is unknown, but it is thought that it can play a role in repair between DNA strands because of the structural similarity with Drosophila Polθ. The Polθ gene is expressed in immune system tissues such as lymph nodes, thymus, bone marrow, fetal liver and tonsils, non-immune tissues such as colon, testis and fetal brain, and B cells such as GC B cells. It is known (see Non-Patent Document 1).

To date, there are several reports on the relationship between the Polθ gene and diseases.
For example, Non-Patent Document 1 describes that the expression of the Polθ gene is enhanced in various cancer tissues, and abnormal expression can be involved in tumor deterioration.
However, nothing is known about the relationship between the Polθ gene and immunodeficiency diseases.
Kawamura et al., International Journal of Cancer 109: 9-16 (2004)

  Functional analysis of genes leads to the development of drugs or reagents having new action mechanisms for various diseases. The present invention provides a drug or a reagent having a new action mechanism for various diseases based on the knowledge obtained by the functional analysis of the Polθ gene, and a useful means for developing a drug or a reagent. The purpose is to provide. Another object of the present invention is to provide a useful means for the functional analysis of the Polθ gene itself.

  The present inventor produced Polθ knockout (KO) mice and analyzed their traits, and found that the number of B cells was reduced in the mice. Therefore, it is considered that the Polθ gene can play an important role in immune function. In addition, it is considered that animals and cells lacking the function of the Polθ gene may be useful for the development of drugs and research reagents for diseases such as immune diseases, and for research uses such as analysis of the Polθ gene or immune function regulation mechanism. It is done.

Based on the above, the present inventors have completed the present invention. That is, the present invention is as follows:
[1] A non-human animal containing a functional defect of the Polθ gene;
[2] The animal according to [1] above, which is a transgenic animal;
[3] The animal according to [1] above, wherein the number of B cells is reduced;
[4] The animal according to the above [1], which is an immunodeficiency disease model;
[5] The animal according to [4] above, wherein the immunodeficiency disease is a disease accompanied by suppression of humoral immune function;
[6] A method for producing a non-human chimeric animal containing a functional defect of the Polθ gene, comprising the following steps (a) to (c):
(A) providing an embryonic stem cell containing a functional defect of the Polθ gene;
(B) introducing the embryonic stem cell into an embryo to obtain a chimeric embryo;
(C) transferring the chimeric embryo to an animal to obtain a chimeric animal;
[7] A method for producing a non-human transgenic animal containing a functional defect of the Polθ gene, comprising the following steps (a) to (d):
(A) providing an embryonic stem cell containing a functional defect of the Polθ gene;
(B) introducing the embryonic stem cell into an embryo to obtain a chimeric embryo;
(C) transferring the chimeric embryo to an animal to obtain a chimeric animal;
(D) mating the chimeric animal to obtain a Polθ gene-deficient heterozygote;
[8] The method according to [7] above, further comprising the step of mating a Polθ gene-deficient heterozygote to obtain a Polθ gene-deficient homozygote;
[9] An animal cell containing a functional defect of the Polθ gene;
[10] The animal cell of [9], which is a transgenic cell;
[11] The animal cell according to [10], which is a cell derived from a non-human transgenic animal containing a functional defect of the Polθ gene;
[12] The animal cell of [9], which is an embryonic stem cell;
[13] The animal cell of [9], which is a B cell;
[14] A method for producing an animal cell containing a functional defect of the Polθ gene, comprising the following steps (a) to (c):
(A) providing a targeting vector capable of inducing homologous recombination of Polθ gene;
(B) introducing the targeting vector into animal cells;
(C) selecting a cell that has undergone homologous recombination from animal cells into which the targeting vector has been introduced;
[15] A method for producing an animal cell containing a functional defect of the Polθ gene, comprising the step of isolating cells from a non-human transgenic animal containing a functional defect of the Polθ gene;
[16] A targeting vector capable of inducing homologous recombination of the Polθ gene, comprising a first polynucleotide and a second polynucleotide homologous to the Polθ gene, and a selectable marker;
[17] A method for producing a targeting vector capable of inducing homologous recombination of the Polθ gene, comprising inserting a first polynucleotide and a second polynucleotide homologous to the Polθ gene, and a selection marker into the vector;
[18] A composition for regulating immune function, comprising a substance that regulates the expression or function of Polθ gene;
[19] The composition according to [18] above, which is a regulator of B cell number;
[20] The composition according to [18] above, which comprises a substance that promotes the expression or function of the Polθ gene;
[21] The composition of [20] above, wherein the substance that promotes the expression or function of Polθ gene is Polθ protein or an expression vector comprising a nucleic acid encoding Polθ protein;
[22] The composition according to [18] above, comprising a substance that suppresses the expression or function of the Polθ gene;
[23] The composition according to [22] above, wherein the substance that suppresses the expression or function of the Polθ gene is any one of (i) and (ii) below:
(I) antisense nucleic acid, ribozyme or RNAi-inducible nucleic acid molecule, or an expression vector comprising these;
(Ii) an antibody or a dominant negative mutant, or an expression vector comprising a nucleic acid encoding them;
[24] The composition of the above-mentioned [18], which is a medicine;
[25] The composition according to [24] above, which is a prophylactic / therapeutic agent for immunodeficiency diseases;
[26] The composition of [25] above, wherein the immunodeficiency disease is a disease accompanied by suppression of humoral immune function;
[27] A screening method for a substance capable of regulating immune function, comprising evaluating whether a test substance can regulate the expression or function of Polθ gene;
[28] The method of [27] above, wherein the substance capable of regulating immune function is a substance capable of regulating the number of B cells;
[29] The method according to [27] above, comprising evaluating whether the test substance can promote the expression or function of the Polθ gene;
[30] The method according to [27] above, comprising evaluating whether or not the test substance can suppress the expression or function of the Polθ gene;
[31] The method of the above [27], comprising the following steps (a) to (c):
(A) contacting the test substance with a cell capable of measuring the expression of the Polθ gene;
(B) measuring the expression level of the Polθ gene in a cell contacted with the test substance, and comparing the expression level with the expression level of the Polθ gene in a control cell not contacted with the test substance;
(C) a step of selecting a test substance that regulates the expression level of the Polθ gene based on the comparison result of (b) above;
[32] The method according to [27] above, comprising the following steps (a) to (c):
(A) contacting the test substance with Polθ protein;
(B) a step of measuring the binding ability of the test substance to the Polθ protein;
(C) a step of selecting a test substance capable of binding to the Polθ protein based on the result of (b) above;
[33] The method of [27] above, which is a method for screening a medicine;
[34] The method of [33] above, which is a screening method for an agent for preventing / treating an immunodeficiency disease;
[35] The method of [34] above, wherein the immunodeficiency disease is a disease accompanied by suppression of humoral immune function;
[36] A method for identifying a Polθ gene polymorphism that causes a change in the ability to regulate immune function, comprising analyzing whether a specific polymorphism in the Polθ gene causes a change in ability to regulate immune function;
[37] A method for determining the onset or risk of developing an immune disease in an animal, comprising measuring the expression level of the Polθ gene using a biological sample of the animal;
[38] A diagnostic agent for the onset or risk of developing an immune disease, comprising a reagent for measuring the expression level of the Polθ gene;
[39] A method for determining the risk of developing an immune disease in an animal, comprising measuring a polymorphism of the Polθ gene using a biological sample of the animal;
[40] A diagnostic agent for the risk of developing an immune disease, comprising a reagent for measuring a polymorphism of the Polθ gene.

  The animals and cells of the present invention are useful as model animals and cells for immunodeficiency diseases, and for analysis of Polθ genes and immune function regulation mechanisms. The targeting vector of the present invention is useful for producing the animal and animal cell of the present invention. The composition of the present invention is useful as a medicine and a research reagent for immune diseases such as immunodeficiency diseases, autoimmune diseases, and allergic diseases. The screening method of the present invention is useful for development of drugs and research reagents for immune diseases. The determination method and diagnostic agent of the present invention are useful for evaluating the onset or risk of developing immune diseases.

(1. animals)
The present invention provides a non-human animal containing a functional defect of the Polθ gene.

  The species of the animal of the present invention is not particularly limited as long as it is an animal excluding humans, but mammals and birds are preferred. Mammals include, for example, laboratory animals such as mice, rats, hamsters, guinea pigs and rabbits, domestic animals such as pigs, cows, goats, horses and sheep, pets such as dogs and cats, primates such as monkeys, orangutans and chimpanzees. Is mentioned. Examples of birds include chickens.

  The functional defect of the Polθ gene refers to a state in which the normal function inherent to the Polθ gene cannot be sufficiently exerted, for example, the state in which the Polθ gene is not expressed at all, or the normal function inherent to the Polθ gene cannot be exhibited. A state in which the expression level is reduced to a certain extent, a state in which the function of the Polθ gene product is completely lost, or a state in which the function of the Polθ gene product is reduced to such an extent that the normal function inherent in the Polθ gene cannot be exhibited. It is done.

As used herein, a B cell can be any B-lineage cell, eg, a pro-B cell, pre-B found in the bone marrow, depending on the stage of differentiation. It can be classified into cells, immature B cells, mature B cells found in the spleen, GC (germinal center) B cells, and memory B cells and plasma cells based on their properties. Since cell markers specific to each cell are known, those skilled in the art can identify the type of B cell by an immunological technique, and by using a cell sorting method such as FACS. , B cells can be sorted according to their type. For example, in mice, the cell markers are as follows: B cells (B220 ⁺ ); pro B cells (B220 ⁺ , CD43 ⁺ , IgM ⁻ ), pre-B cells (B220 ⁺ , CD43 ⁻ , IgM ⁻ ); Immature B cells (B220 ⁺ , CD43 ⁻ , IgM ⁺ ); mature B cells (B220 ⁺ , PNA ⁻ ); GC B cells (B220 ⁺ , PNA ⁺ ).

  The animal of the present invention has various characteristics associated with a functional defect of the Polθ gene. For example, the number of B cells (for example, pre-B cells, immature B cells, mature B cells) in a living body, for example, bone marrow, can be reduced in the animals of the present invention compared to wild-type animals.

The animals of the present invention may also have B cells that have specifically reduced proliferation responsiveness to antigen stimulation compared to B cells derived from wild-type animals. As used herein, an antigen means a substance (eg, an exogenous substance, an endogenous substance) that can act as an antigen or antigen mimic on a B cell, such as an antigen on a B cell. Examples of substances that can act as biomolecules (eg, polypeptides), microorganisms (eg, bacteria, fungi), etc., and substances that can act as antigen mimics on B cells include anti-IgM antibodies Is mentioned. The proliferation responsiveness of B cells can be measured, for example, by evaluating the uptake of [ ³ H] thymidine (for example, see Reference Example 2 described later).

  The animals of the present invention may further have B cells with a reduced survival rate compared to B cells derived from wild type animals. A decrease in B cell viability can be observed in B cells after antigen stimulation. Decreased B cell viability may be due to accelerated apoptosis, a decrease in mitotic (S and G2 / M) cells. The survival rate of B cells can be measured by, for example, a cell cycle assay or propidium iodide staining (for example, see Reference Example 3 described later).

  In one embodiment, the animal of the present invention may be an animal with a genomic DNA modification, a so-called transgenic animal. The transgenic animal of the present invention may be a Polθ gene-deficient heterozygote or a Polθ gene-deficient homozygote.

  The transgenic animals of the present invention can also be animals that contain a functional defect of the cell-specific Polθ gene. Such animals preferably include at least a functional defect of the Polθ gene in B cells. Such animals may exhibit the traits described above in their B cells.

  The transgenic animal of the present invention can be produced by a method known per se. First, a method for producing a chimeric animal useful for producing the transgenic animal of the present invention will be described. The animals of the present invention also include chimeric animals that are useful for producing the transgenic animals of the present invention.

The chimeric animal of the present invention can be produced, for example, by a method including the following steps (a) to (c).
(A) providing an embryonic stem cell containing a functional defect of the Polθ gene;
(B) introducing the embryonic stem cell into an embryo to obtain a chimeric embryo;
(C) transferring the chimeric embryo to an animal to obtain a chimeric animal;

  In step (a) of the above method, embryonic stem cells (ES cells) containing a functional defect of the Polθ gene can be prepared by the method described below, for example.

  In step (b) of the above method, the animal species from which the embryo is derived can be the same as the transgenic animal species of the present invention, and is preferably the same as the animal species from which the embryonic stem cells to be introduced are derived. . Examples of embryos include blastocysts and 8-cell stage embryos. Embryos can be obtained by mating female animals that have undergone superovulation treatment with hormone agents (for example, using PMSG having FSH-like action and hCG having LH action) and the like. Examples of methods for introducing embryonic stem cells into the embryo include micromanipulation methods and aggregation methods.

  In step (c) of the above method, the chimeric embryo can be transferred to the uterus of the animal. The animal into which the chimeric embryo is transferred is preferably a pseudopregnant animal. A pseudopregnant animal can be obtained by mating a female animal having a normal cycle with a male animal castrated by vagina ligation or the like. The animal into which the chimeric embryo has been introduced becomes pregnant and gives birth to the chimeric animal.

  Next, it is confirmed whether the born animal is a chimeric animal. Whether or not the animal born is a chimeric animal can be confirmed by a method known per se, for example, by body color or coat color. For discrimination, DNA may be extracted from a part of the body, and Southern blot analysis or PCR assay may be performed.

The transgenic animal of the present invention can be produced, for example, by a method including the following steps (a) to (d):
(A) providing an embryonic stem cell containing a functional defect of the Polθ gene;
(B) introducing the embryonic stem cell into an embryo to obtain a chimeric embryo;
(C) transferring the chimeric embryo to an animal to obtain a chimeric animal;
(D) A step of mating the chimeric animal to obtain a Polθ gene-deficient heterozygote.

  Steps (a) to (c) of the above method can be performed in the same manner as the method for producing a chimeric animal described above.

  In step (d) of the above method, the chimeric animal obtained in step (c) is mated after it matures. The mating can preferably take place between wild-type animals and chimeric animals or between chimeric animals. Whether or not the Polθ gene deficiency has been introduced into germline cells of a chimeric animal and a Polθ gene deficient heterozygous offspring has been obtained can be confirmed by various methods using indices known per se, for example, the body of a progeny animal. It can be distinguished by color and coat color. For discrimination, DNA may be extracted from a part of the body, and Southern blot analysis or PCR assay may be performed. Furthermore, a Polθ gene-deficient homozygote can be produced by mating the Polθ gene-deficient heterozygotes thus obtained.

  In general, in the process of producing a transgenic animal, a progeny animal having a genotype in which a gene derived from an embryonic stem cell and a gene derived from an animal used for mating are hybridized is obtained. In some cases, it may be difficult to examine the peculiar effect only due to lack of. Therefore, in order to more appropriately extract only the effects peculiar to Polθ gene deficiency, the obtained Polθ gene deficient animal (heterozygote or homozygote) is backcrossed to a pure animal strain for about 5 to 8 generations. It is preferable to do. In addition, when backcrossing is carried out only by natural mating, it may take a long time, so in vitro fertilization techniques can be used as appropriate when it is desired to speed up generational changes.

  In addition, when the transgenic animal of the present invention is a transgenic animal containing a functional defect of a cell-specific Polθ gene, the animal can be obtained, for example, by using a predetermined targeting vector (described later) containing a recombinase target sequence. It can be produced by mating the produced animal with a transgenic animal that expresses recombinase in at least B cells (for example, a transgenic animal that expresses recombinase specifically for B cells). For example, CD19-Cre transgenic mice have been reported as transgenic animals expressing recombinase specifically for B cells (see, for example, Rickert et al., Nucleic Acid Res. 25: 1317-1318 (1997)). . Moreover, you may make a recombinase or a recombinase expression vector act on the B cell of the animal produced by use of the targeting vector (after-mentioned) containing a recombinase target sequence. For example, as a method of causing recombinase to act on B cells, or a method of causing recombinase to act specifically on the cells, a method using an expression vector containing a B cell specific promoter (for example, CD19 promoter), Epstein Examples thereof include a method using bar virus as an expression vector, and a method using a liposome in which an antibody against a cell surface molecule specifically expressed in B cells is incorporated.

  In another embodiment, the animal of the present invention can be an animal without genomic DNA modification. Such an animal can be produced, for example, by forced expression of a substance (for example, an antisense nucleic acid or an RNAi-inducible nucleic acid) that suppresses the expression or function of the Polθ gene described later in the animal. Administration can be carried out using a suitable delivery means such as, for example, liposomes. It is also possible to achieve a cell-specific functional defect of the Polθ gene. For example, in order to achieve B cell-specific functional loss of the Polθ gene, a method similar to the method of allowing recombinase to specifically act on B cells can be used.

  The animal of the present invention is useful, for example, as an immunodeficiency disease model. The immunodeficiency disease that the animal of the present invention can serve as a model can be a disease accompanied by suppression of humoral immune function, that is, a disease that can be caused by a decrease in the number of B cells and / or inactivation of B cells. Examples of the disease accompanied by suppression of humoral immune function include diseases accompanied by a decrease in antibody production ability (eg, γ globulin disease, BLNK deficiency).

  The animal of the present invention is also useful for analysis of Polθ gene and immune function regulating mechanism, and development of immune function regulating drugs. For example, comprehensive analysis of gene expression in the animal of the present invention makes it possible to identify other genes involved in immune function regulation (for example, genes showing an expression pattern linked to the expression pattern of the Polθ gene). . In this case, for example, in the animal of the present invention, the gene expression profile is measured by means (for example, microarray) that enables comprehensive analysis of gene expression, and a control animal (such as a wild type animal or other immune disease model animal) Compared to the gene expression profile of the same or different animals. In addition, the gene expression profile of the animal of the present invention can be traced over time, and the link between the expression and progression of the disease state and the change in the gene expression profile can be evaluated.

(2. Animal cells)
The present invention also provides an animal cell containing a functional defect of the Polθ gene.

  The animal cell of the present invention may be a cell derived from any animal including a human, but a cell derived from a mammal or a bird is preferred. Examples of mammalian species from which the animal cells of the present invention are derived include, for example, laboratory animals such as mice, rats, hamsters, guinea pigs, rabbits, domestic animals such as pigs, cows, goats, horses, sheep, pets such as dogs and cats, Primates such as monkeys, orangutans, chimpanzees and humans. Examples of birds include chickens.

  The animal cell of the present invention can also be a cell derived from any tissue, for example, a somatic cell (eg, B cell) expressing a Polθ gene, a spermatogonia cell, a germ line cell such as a sperm, an egg, And embryonic stem cells. The animal cell of the present invention may be either a primary cultured cell or a cell line.

When the animal cell of the present invention is a B cell, the B cell can exhibit various traits similar to the B cell of the transgenic animal of the present invention. For example, the B cells of the present invention can have a specific decrease in proliferation responsiveness to antigen stimulation, compared to B cells derived from wild-type animals. The proliferation responsiveness of B cells can be measured, for example, by evaluating the uptake of [ ³ H] thymidine (for example, see Reference Example 2 described later).

  In addition, the survival rate of the B cells of the present invention can be reduced as compared to B cells derived from wild-type animals. A decrease in B cell viability can be observed in B cells after antigen stimulation. Decreased B cell viability may be due to accelerated apoptosis, a decrease in mitotic (S and G2 / M) cells. The survival rate of B cells can be measured by, for example, a cell cycle assay or propidium iodide staining (for example, see Reference Example 3 described later).

  In one embodiment, the animal cell of the present invention may be a cell with genomic DNA modification, a so-called transgenic cell. The transgenic cell of the present invention can be, for example, a Polθ gene-deficient homozygote or a Polθ gene-deficient heterozygote.

The transgenic cell of the present invention can be produced by a method known per se. For example, the animal cell of the present invention can be produced by a method including the following steps (a) to (c).
(A) providing a targeting vector capable of inducing homologous recombination of Polθ gene;
(B) introducing the targeting vector into animal cells;
(C) A step of selecting cells that have undergone homologous recombination from cells into which the targeting vector has been introduced.

  In step (a) of the above method, for example, the targeting vector of the present invention (described later) can be used as a targeting vector capable of inducing homologous recombination of the Polθ gene.

  In step (b) of the above method, a targeting vector is introduced into the animal cell. Examples of the method for introducing the targeting vector into animal cells include the calcium phosphate method, the lipofection method / liposome method, and the electroporation method. When the targeting vector is introduced into an animal cell, homologous recombination of genomic DNA containing the Polθ gene occurs in the animal cell.

  As an animal cell into which the targeting vector is introduced, one produced by a method known per se, a commercially available one, or one available from a predetermined organization can be used.

  For example, when an embryonic stem cell is used as an animal cell into which a targeting vector is introduced, the embryonic stem cell can be established by culturing an inner cell mass separated from a blastocyst of any animal on a feeder cell. Although it is good, existing embryonic stem cells can be obtained commercially or from a predetermined organization. Examples of existing mouse embryonic stem cells include ES-D3 cells, ES-E14TG2a cells, SCC-PSA1 cells, TT2 cells, AB-1 cells, J1 cells, R1 cells, E14.1 cells, RW-4 cells and the like. Is mentioned. In addition to mouse embryonic stem cells, embryonic stem cells derived from mammals such as humans, mink, hamsters, pigs, cattle, marmoset, rhesus monkeys, etc. have been established at present. You can also.

  When somatic cells such as B cells are used as animal cells into which the targeting vector is introduced, the somatic cells into which the targeting vector is introduced may be either primary cultured cells or cell lines. Primary cultured cells and cell lines can be prepared by methods known per se (see, for example, Current Protocols in Cell Biology, John Wiley & Sons, Inc. (2001); isolation and culture of functional cells, Maruzen Shoten (1987)).

  In step (c) of the above method, animal cells after introduction of the targeting vector are screened in order to select animal cells in which homologous recombination has occurred with genomic DNA containing the Polθ gene. For example, after selection by positive selection, negative selection, etc., screening based on genotype (for example, PCR method, Southern blot hybridization method) is performed.

  When the animal cell is an embryonic stem cell, preferably a karyotype analysis of the recombinant embryonic stem cell is further performed. Karyotype analysis confirms that there is no chromosomal abnormality in the selected recombinant embryonic stem cells. Karyotype analysis can be performed by a method known per se. The karyotype of embryonic stem cells is preferably confirmed in advance before introducing the targeting vector.

  The transgenic cells of the present invention can also be isolated from the transgenic animals of the present invention. Since the transgenic animal of the present invention contains a functional defect of the Polθ gene, the transgenic cell isolated from the transgenic animal of the present invention also contains a functional defect of the Polθ gene. In addition, cells isolated from the transgenic animal of the present invention may be modified by a method such as a genetic engineering technique. Isolation and modification of cells can be performed by methods known per se (see, for example, Current Protocols in Cell Biology, John Wiley & Sons, Inc. (2001); isolation and culture of functional cells, Maruzen Shoten (1987)). ).

  In another embodiment, the animal cell of the present invention can be a cell without genomic DNA modification. Such a cell can be prepared, for example, by introducing into a cell a substance (for example, an antisense nucleic acid or an RNAi-inducible nucleic acid) that suppresses the expression or function of the Polθ gene described later. The substance that suppresses the expression or function of Polθ gene can be introduced into cells by a method known per se, for example, the substance that suppresses the expression or function of Polθ gene is a nucleic acid molecule or an expression vector containing the nucleic acid molecule. In this case, calcium phosphate method, lipofection method / liposome method, electroporation method and the like are used.

  The animal cell of the present invention is useful for analysis of Polθ gene, immune function regulating mechanism and immunodeficiency disease, and development of immune function regulating drug.

(3. Targeting vector)
The present invention provides a targeting vector capable of inducing homologous recombination of the Polθ gene, comprising a first polynucleotide and a second polynucleotide homologous to the Polθ gene, and a selectable marker.

  The first and second polynucleotides are polynucleotides having a sufficient degree of sequence identity and length to cause homologous recombination with the genomic DNA containing the Polθ gene. The first and second polynucleotides each correspond to a different region of genomic DNA containing the Polθ gene.

  Also, the first and second polynucleotides are selected so as to cause a functional defect of the Polθ gene by homologous recombination. That is, the first and second polynucleotides in the genomic DNA containing the Polθ gene, when a genomic DNA partial region existing between two regions homologous to the first and second polynucleotides is deleted, Selected to result in a functional defect in the Polθ gene. Such a region can be appropriately determined by those skilled in the art. For example, the first and second polynucleotides are selected so that one or more exons or at least a part of the promoter region or enhancer region is deleted. In addition, the first and second polynucleotides are selected so that at least a part of exons corresponding to the helicase domain (exons 2 to 9) or exons corresponding to the polymerase domain (for example, exons 20 to 29) is deleted. It is also preferable to do.

For example, in mice, each exon of the Polθ gene corresponds to the following position in the nucleotide sequence shown in SEQ ID NO: 1:
Exon 1: Ex1 to 274 Exon 2: 1322 to 1501 Exon 3: 3294 to 3424 Exon 4: 5414 to 5570 Exon 5: 10949 to 11057 Exon 6: 15985 to 16204 Exon 7 : 17558th to 17705th exon 8: 21607th to 21753th exon 9: 23012th to 23224th exon 10: 28701th to 28837th exon 11: 29940th to 30144th exon 12: 30332th to 30474th exon 13: 32956 Th to 33149th exon 14: 33968th to 34092th exon 15: 36758th to 37001th exon 16: 48997th to 51977th ex 17: 54266th to 54409th exon 18: 55774th to 55970th exon 19: 60582th to 60822th exon 20: 62916th to 63109th exon 21: 66027th to 66164th exon 22: 67297th to 67471st exon 23 71037th to 71160th exon 24: 71725th to 71846th exon 25: 75703th to 75887th exon 26: 78465th to 78576th exon 27: 80226th to 80350th exon 28: 82017th to 82170th exon 29: 83209 Th to 83324 th exon 30 (last exon): 83696 th to 84592 th

  The degree of identity of the first and second polynucleotides to the genomic DNA containing the Polθ gene is not particularly limited as long as homologous recombination is possible. For example, the degree of identity that allows homologous recombination depends on the length of the polynucleotide, but for example at least about 80% or more, preferably at least about 85% or more, more preferably at least about 90% or more, most Preferably it may be about 95-100%. The percent identity can be determined by a method known per se, for example, by using BLASTN available on the NCBI homepage with default settings. % Identity also uses the Smith-Waterman algorithm to affine gap search with a gap open penalty of 12 and a gap extension penalty of 1. ) May be determined.

  The length of the first and second polynucleotides is not particularly limited as long as homologous recombination of genomic DNA occurs. However, in general, a longer homologous region is better for efficient homologous recombination of genomic DNA with a targeting vector. On the other hand, the length of DNA that can be inserted is limited to a certain value depending on the type of targeting vector. Therefore, considering these, the length of the first polynucleotide and the second polynucleotide can be, for example, 0.5 kb-20 kb, preferably 1 kb-10 kb.

  In one embodiment, a selectable marker is included between the first polynucleotide homologous to the Polθ gene and the second polynucleotide (in other words, inside). In this case, the selection marker is preferably a positive selection marker. A positive selectable marker is a gene that encodes a product that allows only cells having the gene to survive and / or grow under predetermined conditions, such as a neomycin resistance gene, a hygromycin B phosphotransferase (BPH) gene, a blast Examples include the shidine S deaminase gene and the puromycin resistance gene.

  In another embodiment, a selectable marker is included outside the first polynucleotide homologous to the Polθ gene and the second polynucleotide. In this case, the selection marker is preferably a negative selection marker. Negative selectable markers are genes that act toxic to cells having DNA inserts integrated into non-targeting chromosomal sites, such as the herpes simplex virus (HSV) thymidine kinase (tk) gene, diphtheria toxin A fragment (DTA) gene etc. are mentioned.

  The targeting vector of the present invention can contain either a positive selection marker or a negative selection marker, preferably both.

  The vector serving as the basic skeleton of the targeting vector of the present invention is not particularly limited as long as it can replicate itself in a cell to be transformed (for example, E. coli). For example, commercially available pBluescript (manufactured by Stratagene), pZErO 1.1 (Invitrogen), pGEM-1 (Promega) and the like can be used.

  The targeting vector of the present invention may also contain two or more recombinase target sequences. Two or more recombinase target sequences can be placed in the same or opposite orientation. Two or more recombinase target sequences are located between the first and second polynucleotides homologous to the Polθ gene (in other words, inside). Such targeting vectors allow so-called conditional knockouts.

  As a recombinase target sequence, a sequence known in the art, for example, a loxP sequence used in a Cre / loxP system derived from bacteriophage P1, or an FRT sequence used in a FLP / FRT system derived from yeast can be used.

  In one embodiment, a partial region in the genomic DNA containing the Polθ gene or the region and a positive selection marker are placed between two recombinase target sequences (in other words, inside). Specifically, the recombinase target sequence is arranged in the form of a recombinase target sequence—partial region (and positive selection marker) in genomic DNA containing the Polθ gene—recombinase target sequence.

  In another embodiment, a partial region in the genomic DNA containing the Polθ gene and a positive selection marker are individually placed between the three recombinase target sequences. Specifically, the recombinase target sequence is arranged in the form of recombinase target sequence-partial region in genomic DNA containing Pol.theta. Gene-recombinase target sequence-positive selection marker-recombinase target sequence.

  A partial region in the genomic DNA contained between two or more recombinase target sequences is a region in which functional deletion of the Polθ gene is caused by deletion of the region under the action of recombinase. Such a region can be appropriately determined by those skilled in the art, and may be, for example, a region including at least one exon, or a promoter region or an enhancer region.

  The targeting vector of the present invention can be produced by a method known per se. For example, the targeting vector of the present invention can be produced by inserting a first polynucleotide and a second polynucleotide homologous to the Polθ gene and the selection marker into the vector. In order to prepare such a targeting vector, it is necessary to first isolate a genomic DNA fragment containing the Polθ gene. However, the genomic DNA fragment may be efficiently recombined during homologous recombination. It is preferable to isolate from the same animal species from which the ES cells to be produced are derived. In order to further increase the efficiency of homologous recombination, it is more preferable to isolate genomic DNA from animals of the same strain among animals of the same species from which ES cells are derived.

  The targeting vector of the present invention is useful, for example, for producing the animal and animal cell of the present invention.

For details of the production of the animal, animal cell, and targeting vector of the present invention, see, for example, the following documents.
1. Separate Experiment Medicine The Protocol Series “The Latest Technology of Gene Turding” (2000, Yodosha) Conditional Targeting Method p.115-120
2. Biomanual Series 8 “Gene Targeting”-Production of mutant mice using ES cells (1995, Yodosha) p.71-77
3. Sambrook et al., Molecular Cloning: A LABORATORY MANUAL, 3rd edition, COLD SPRING HARBO
R LABORATORY PRESS, 2001, 4. 82-4.85
4). Robertson EJ in Teratocarcinomas and embryonic stem cells-a practical approach, ed. Robertson, EJ (IRL Press, Oxford), 1987: pp.108-112
5. Dynecki, SM et al., Gene Targeting -a practical approach, 2nd edition, ed. Joyner, AL (Oxford Univ. Press), 2000: pp.68-73
6). Dynecki, SM et al., Gene Targeting -a practical approach, 2nd edition, ed.Joyner, AL (Oxford Univ. Press), 2000: pp.75-81

(4. Composition for regulating immune function)
The present invention also provides a composition for regulating immune function, comprising a substance that regulates the expression or function of Polθ gene.

  In one embodiment, the substance that modulates Polθ gene expression or function may be a substance that promotes Polθ gene expression.

  The expression of the Polθ gene means that a translation product (ie, protein) from the Polθ gene is produced and functionally localized at the site of action. Therefore, the substance that promotes the expression of the Polθ gene may act at any stage such as transcription of the Polθ gene, post-transcriptional regulation, translation, post-translational modification, localization, and protein folding. As used herein, promotion of Polθ gene expression includes supplementation of Polθ protein.

  An example of a substance that promotes the expression of the Polθ gene can be a Polθ protein or an expression vector containing a nucleic acid encoding the Polθ gene.

  In another embodiment, the substance that modulates Polθ gene expression or function may be a substance that suppresses Polθ gene expression. The substance that suppresses the expression of the Polθ gene may act at any stage such as transcription, post-transcriptional regulation, translation, post-translational modification, localization, and protein folding of the Polθ gene.

  An example of a substance that suppresses the expression of the Polθ gene is an antisense nucleic acid against a transcription product of the Polθ gene, specifically, mRNA or an initial transcription product. The antisense nucleic acid is composed of a base sequence capable of hybridizing with the target mRNA (initial transcript) under physiological conditions of a cell expressing the target mRNA (initial transcript), and the target mRNA in the hybridized state. A nucleic acid capable of inhibiting translation of a polypeptide encoded by (early transcript). The type of the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. In addition, since the phosphodiester bond of a natural type antisense nucleic acid is easily degraded by a nucleolytic enzyme present in cells, the antisense nucleic acid of the present invention has a thiophosphate type (phosphorus) that is stable to the degrading enzyme. It is also possible to synthesize using modified nucleotides such as P′O of acid bond and P═S) or 2′-O-methyl type. Other important factors for the design of antisense nucleic acid include enhancement of water solubility and cell membrane permeability. These can also be overcome by devising dosage forms such as the use of liposomes and microspheres.

  The length of the antisense nucleic acid is not particularly limited as long as it can specifically hybridize with the transcription product of the Polθ gene. The short one is about 15 bases, and the long one is complementary to the entire sequence of mRNA (initial transcription product). The sequence may include a sequence. From the viewpoint of ease of synthesis, antigenicity problems, etc., for example, oligonucleotides comprising about 15 bases or more, preferably about 15 to about 30 bases are exemplified.

  The target sequence of the antisense nucleic acid is not particularly limited as long as the antisense nucleic acid hybridizes to inhibit the translation of the Polθ gene or a functional fragment thereof, and even a partial sequence of mRNA is a partial sequence. Or an intron portion of the initial transcript, but when an oligonucleotide is used as the antisense nucleic acid, the target sequence is from the 5 ′ end of the Polθ gene mRNA to the C-terminus of the coding region. It is desirable to be located by.

  Furthermore, antisense nucleic acids not only hybridize with the transcription product of the Polθ gene and inhibit translation, but also bind to double-stranded DNA to form a triplex and inhibit transcription into mRNA. It may be obtained.

  Another example of a substance that suppresses expression of the Polθ gene is a Polθ gene transcript, specifically mRNA or an initial transcript, specifically within the coding region (including an intron in the case of the initial transcript). Ribozymes that can be cleaved. Ribozyme refers to RNA having an enzyme activity that cleaves nucleic acid. Recently, it has been clarified that an oligo DNA having a base sequence of the enzyme active site also has a nucleic acid cleaving activity. As long as it has specific nucleic acid cleavage activity, it is used as a concept including DNA. The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and the hammerhead type and hairpin type are known. Further, when the ribozyme is used in the form of an expression vector containing DNA encoding the ribozyme, it can be a hybrid ribozyme in which a sequence modified with tRNA is further linked in order to promote the transfer to the cytoplasm [ Nucleic Acids Res., 29 (13): 2780-2788 (2001)].

  Yet another example of a substance that suppresses the expression of the Polθ gene is an RNAi-inducible nucleic acid. An RNAi-inducible nucleic acid refers to a nucleic acid that can induce an RNAi effect when introduced into a cell, and is preferably RNA. The RNAi effect refers to a phenomenon in which RNA having a double-stranded structure containing the same nucleotide sequence (or a partial sequence thereof) as mRNA suppresses the expression of the mRNA. In order to obtain the RNAi effect, for example, it is preferable to use RNA having a double-stranded structure having at least 20 or more consecutive target mRNAs and the same nucleotide sequence (or a partial sequence thereof). The double-stranded structure may be composed of different strands, or may be a double-stranded chain provided by a single RNA stem-loop structure. Examples of the RNAi-inducible nucleic acid include siRNA, stRNA, miRNA and the like. As the RNAi-inducible nucleic acid, one synthesized by itself can be used as described later, but a commercially available one may be used (for example, available from stealth RNAi (manufactured by Invitrogen, catalog number: 10620-310)).

  Antisense oligonucleotides and ribozymes, for example, determine the transcription product of Polθ gene based on the cDNA sequence or genomic DNA sequence of Polθ gene, specifically mRNA or the initial transcription product target sequence, and commercially available DNA / RNA automatic synthesis It can be prepared by synthesizing a complementary sequence using a machine (Applied Biosystems, Beckman, etc.). For decoy nucleic acid and RNAi-inducible nucleic acid, for example, the sense strand and the antisense strand were respectively synthesized by a DNA / RNA automatic synthesizer and denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer. Thereafter, it can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. In addition, longer double-stranded polynucleotides can be prepared by synthesizing complementary oligonucleotide strands so as to alternately overlap, annealing them, and then ligating with ligase.

Another example of a substance that suppresses the expression of the Polθ gene is an antibody against the Polθ protein. The antibody may be a polyclonal antibody or a monoclonal antibody, and can be prepared by a known immunological technique. The antibody may be an antibody fragment (eg, Fab, F (ab ′) ₂ ) or a recombinant antibody (eg, a single chain antibody). Furthermore, a nucleic acid encoding the antibody (that is operably linked to a nucleic acid having promoter activity) is also preferable as a substance that suppresses the expression of the Polθ gene.

  For example, a polyclonal antibody may be a commercially available adjuvant (eg, a complex cross-linked to a carrier protein such as bovine serum albumin or KLH (Keyhole Limpet Hemocyanin), if necessary. For example, complete or incomplete Freund's adjuvant) is administered to the animal subcutaneously or intraperitoneally about 2 to 4 times every 2 to 3 weeks (the antibody titer of the partially collected serum is measured by a known antigen-antibody reaction, It can be obtained by collecting whole blood about 3 to about 10 days after the final immunization and purifying the antiserum. Examples of animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, guinea pigs, and hamsters.

  Monoclonal antibodies can be obtained by cell fusion methods (for example, Takeshi Watanabe, Principles of Cell Fusion Methods and Production of Monoclonal Antibodies, Akira Taniuchi, Toshitada Takahashi, “Monoclonal Antibodies and Cancer: Basic and Clinical”, 2-14). Page, Science Forum Publishing, 1985). For example, the factor is administered to a mouse subcutaneously or intraperitoneally 2-4 times together with a commercially available adjuvant, and about 3 days after the final administration, the spleen or lymph node is collected and white blood cells are collected. The leukocytes and myeloma cells (for example, NS-1, P3X63Ag8, etc.) are fused to obtain a hybridoma that produces a monoclonal antibody against the factor. The cell fusion may be PEG method [J. Immunol. Methods, 81 (2): 223-228 (1985)] or voltage pulse method [Hybridoma, 7 (6): 627-633 (1988)]. A hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a well-known EIA or RIA method. The hybridoma producing the monoclonal antibody can be cultured in vitro or in vivo, such as mouse or rat, preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively.

  However, considering the therapeutic effect and safety in humans, the antibody of the present invention may be a chimeric antibody, a humanized or humanized antibody. Chimeric antibodies include, for example, “Experimental Medicine (Special Issue), Vol. 6, No. 10, 1988”, Japanese Patent Publication No. 3-73280, and humanized antibodies include, for example, Japanese Patent Publication No. 4-506458, JP-A-62-296890 and the like, human antibodies include, for example, `` Nature Genetics, Vol.15, p.146-156, 1997 '', `` Nature Genetics, Vol.7, p.13-21, 1994 '', JP 4-504365 Publication, International Application Publication No. WO 94/25585 Publication, “Nikkei Science, June, 40 to 50, 1995”, “Nature, Vol.368, p.856-859, 1994 ", Japanese Patent Publication No. 6-500233, and the like.

  In yet another embodiment, the substance that regulates the expression or function of the Polθ gene may be a substance that suppresses the function of the Polθ gene. The substance that suppresses the function of the Polθ gene is not particularly limited as long as it is a substance that can interfere with the action of the Polθ protein. Examples include a dominant negative mutant of the Polθ protein and an expression vector containing a nucleic acid encoding the mutant. .

  The dominant negative mutant of Polθ protein refers to one whose activity is reduced by introducing a mutation into Polθ protein. The dominant negative mutant can indirectly inhibit its activity by competing with the natural Polθ protein. The dominant negative mutant can be prepared by introducing a mutation into a nucleic acid encoding the Polθ gene. Examples of the mutation include an amino acid mutation (for example, deletion, substitution, or addition of one or more amino acids) that causes a decrease in the function of the functional site. The dominant negative mutant can be prepared by a method known per se using PCR or a known kit.

  When the substance that regulates the expression or function of the Polθ gene is a nucleic acid molecule or a protein molecule, the composition of the present invention can also contain an expression vector containing a nucleic acid molecule encoding the nucleic acid molecule or protein molecule as an active ingredient. . In the expression vector, the oligonucleotide or polynucleotide encoding the nucleic acid molecule must be operably linked to a promoter capable of exhibiting promoter activity in a mammalian cell to be administered. The promoter to be used is not particularly limited as long as it can function in the mammal to be administered. For example, SV40-derived early promoter, cytomegalovirus LTR, Rous sarcoma virus LTR, MoMuLV-derived LTR, adenovirus-derived early promoter Examples include viral promoters such as promoters, and mammalian constituent protein gene promoters such as β-actin gene promoters, PGK gene promoters, transferrin gene promoters, and the like.

  The expression vector preferably contains a transcription termination signal, ie a terminator region, downstream of the oligo (poly) nucleotide encoding the nucleic acid molecule. In addition, selectable marker genes for selection of transformed cells (such as genes that confer resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, genes that complement auxotrophic mutations, etc.) You can also

  The vector may be a basic skeleton vector, a plasmid or a viral vector used as an expression vector, but suitable vectors for administration to mammals such as humans include adenovirus, retrovirus, adeno-associated virus, herpes virus, vaccinia virus. And viral vectors such as poxvirus, poliovirus, Sindbis virus, Sendai virus, Epstein-Barr virus, and the like.

  The composition of the present invention may be allowed to act specifically on B cells or non-specifically. For example, when a substance that regulates the expression or function of Polθ gene is used in the form of an expression vector, it can be made to act specifically on B cells by using a vector in which a nucleic acid molecule is linked to a B cell specific promoter. (See, for example, “(1. Animals)”).

  The composition of the present invention can contain any carrier, for example, a pharmaceutically acceptable carrier, in addition to the substance that regulates the expression or function of the Polθ gene.

  Examples of pharmaceutically acceptable carriers include sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate and other excipients, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone. , Gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate , Aerosil, Talc, Sodium lauryl sulfate lubricant, Citric acid, Menthol, Glycyllysine / Ammonium salt, Glycine, Orange powder, Fragrance, Sodium benzoate Preservatives such as lithium, sodium hydrogen sulfite, methyl paraben, propyl paraben, stabilizers such as citric acid, sodium citrate, acetic acid, suspensions such as methyl cellulose, polyvinyl pyrrolidone, aluminum stearate, dispersants such as surfactants, Examples include, but are not limited to, water, physiological saline, diluents such as orange juice, base waxes such as cacao butter, polyethylene glycol, and white kerosene.

  Preparations suitable for oral administration include solutions in which an effective amount of a substance is dissolved in a diluent such as water or physiological saline, capsules, sachets or tablets containing an effective amount of the substance as a solid or granule. Examples thereof include a suspension in which an effective amount of a substance is suspended in a suitable dispersion medium, and an emulsion in which a solution in which an effective amount of a substance is dissolved is dispersed in an appropriate dispersion medium and emulsified.

  Formulations suitable for parenteral administration (eg, subcutaneous injection, intramuscular injection, local infusion, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained. Aqueous and non-aqueous sterile suspensions are also included, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials. In addition, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.

  The dose of the composition of the present invention varies depending on the activity and type of the active ingredient, the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, body weight, age, etc. The amount of active ingredient per day for an adult is about 0.001 to about 500 mg / kg.

  The composition of the present invention is useful, for example, as a pharmaceutical or research reagent. For example, when the composition of the present invention is a pharmaceutical composition, it can be used as a prophylactic / therapeutic agent for immune diseases such as immunodeficiency diseases, autoimmune diseases, and allergic diseases.

  Specifically, when the pharmaceutical composition of the present invention contains a substance that promotes the expression or function of the Polθ gene as an active ingredient, for example, as an immunodeficiency disease, a disease involving suppression of immune function, for example, humoral immune function It can be used for the prevention and treatment of Diseases associated with suppression of humoral immune function (for example, immunodeficiency diseases) are as described above (for example, see the section “(1. Animals)”).

  In addition, when the pharmaceutical composition of the present invention contains a substance that suppresses the expression or function of the Polθ gene as an active ingredient, for example, prevention / treatment of diseases accompanied by enhanced humoral immune function as autoimmune diseases or allergic diseases Can be used. A disease associated with enhanced humoral immune function can be a disease that can be caused by an increase in the number of B cells and / or excessive activation of B cells. Examples of autoimmune diseases with enhanced humoral immune function to which the pharmaceutical composition of the present invention can be applied include systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis , Hashimoto's disease, autoimmune hemolytic anemia, pernicious anemia, autoimmune thrombocytopenia, autoimmune cytopenia, myasthenia gravis, scleroderma, polymyositis, secondary Addison's disease, infertility, self Examples include immune glomerulonephritis, Sjogren's disease, and vasculitis. Examples of allergic diseases accompanied by enhanced humoral immune function include type I allergic diseases (eg bronchial asthma, allergic rhinitis, urticaria, atopic dermatitis), type II allergic diseases (eg Hashimoto's disease (chronic) Thyroiditis), Goodpasture syndrome, organ transplant rejection), type III allergic diseases (for example, systemic vasculitis, cryoglobulinemia, glomerulonephritis).

  Furthermore, when the composition of the present invention is a research reagent, it can be used, for example, as an inducer of immune diseases in experimental animals. Specifically, when the research reagent of the present invention contains a substance that promotes the expression or function of the Polθ gene as an active ingredient, it can be used, for example, for induction of immunodeficiency diseases.

  The compositions of the present invention are also useful as regulators of B cell count. Specifically, when the modulator of the present invention contains a substance that promotes the expression or function of the Polθ gene as an active ingredient, it can be used to increase the number of B cells. Moreover, when the regulator of this invention contains the substance which suppresses the expression or function of Pol (theta) gene as an active ingredient, it can be used for the fall of B cell number. When the modulator of the present invention is used with the intention of increasing the number of B cells, B cell proliferation can be further promoted by antigen stimulation. Therefore, in this case, the regulator of the present invention may have both a substance that promotes the expression or function of the Polθ gene and an antigen for B cells, and is a form in which both are combined (for example, the same container Or stored in separate forms (eg, stored in different containers). Note that the modulator of the present invention can be used in vivo or in vitro.

(5. Screening method for substances capable of regulating immune function)
The present invention also includes a method for screening a substance capable of regulating immune function, comprising evaluating whether a test substance can regulate the expression or function of Polθ gene, and a substance obtained by the screening method, and A composition for regulating immune function comprising a substance is provided.

  The test substance to be subjected to the screening method may be any known compound or novel compound, for example, a nucleic acid, carbohydrate, lipid, protein, peptide, low molecular organic compound, compound prepared using combinatorial chemistry technology Examples include libraries, random peptide libraries prepared by solid phase synthesis and phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.

In one embodiment, the screening method of the present invention includes the following steps (a) to (c):
(A) contacting the test substance with a cell capable of measuring the expression of the Polθ gene;
(B) measuring the expression level of the Polθ gene in a cell contacted with the test substance, and comparing the expression level with the expression level of the Polθ gene in a control cell not contacted with the test substance;
(C) A step of selecting a test substance that regulates the expression level of the Polθ gene based on the comparison result of (b).

  In step (a) of the above method, the test substance is placed in contact with cells capable of measuring Polθ gene expression. Contact of the test substance with a cell capable of measuring the expression of the Polθ gene can be performed in a culture medium.

  The cell capable of measuring the expression of the Polθ gene refers to a cell capable of directly or indirectly evaluating the expression level of a product of the Polθ gene, for example, a transcription product or a translation product. A cell capable of directly evaluating the expression level of the Polθ gene product may be a cell capable of naturally expressing the Polθ gene, while a cell capable of indirectly evaluating the expression level of the Polθ gene product may be a Polθ gene. It can be a cell that allows a reporter assay for the gene transcription regulatory region. The cell capable of measuring the expression of the Polθ gene can be an animal cell, for example, a mammalian cell such as a mouse, rat, hamster, guinea pig, rabbit, dog, monkey or human.

  The cell capable of naturally expressing the Polθ gene is not particularly limited as long as it can potentially express the Polθ gene. Such cells can be easily identified by those skilled in the art, and primary cultured cells, cell lines derived from the primary cultured cells, commercially available cell lines, cell lines available from cell banks, and the like can be used. Examples of cell lines in which the Polθ gene is expressed include A20 and 70Z / 3. In addition, since it is known that Pol (theta) gene is expressing in the above-mentioned tissue or cell, you may use the cell or cell line derived from such a tissue or cell (for example, B cell).

  A cell enabling a reporter assay for the Polθ gene transcription regulatory region is a cell comprising a Polθ gene transcriptional regulatory region and a reporter gene operably linked to the region. The Polθ gene transcription regulatory region and the reporter gene can be inserted into an expression vector. The Polθ gene transcription regulatory region is not particularly limited as long as it is a region capable of controlling the expression of the Polθ gene. Examples include a region consisting of a lost, substituted or added base sequence and having the ability to control the transcription of the Polθ gene. The reporter gene may be any gene that encodes a detectable protein or an enzyme that produces a detectable substance. For example, the GFP (green fluorescent protein) gene, GUS (β-glucuronidase) gene, LUC (luciferase) gene, CAT (Chloramphenicol acetyltransferase) gene and the like.

  A cell into which a Polθ gene transcription regulatory region and a reporter gene operably linked to the region are introduced can quantitatively analyze the expression level of the reporter gene as long as the Polθ gene transcription regulatory function can be evaluated. There is no particular limitation. However, since a physiological transcriptional regulatory factor for the Polθ gene is expressed and considered to be more appropriate for evaluating the regulation of the expression of the Polθ gene, the cells to be introduced include cells that can naturally express the Polθ gene. preferable.

  The culture medium in which the test substance is contacted with the cells capable of measuring the expression of the Polθ gene is appropriately selected according to the type of the cells used, but is, for example, a minimum containing about 5 to 20% fetal calf serum. Essential medium (MEM), Dulbecco's modified minimal essential medium (DMEM), RPMI 1640 medium, 199 medium, and the like. The culture conditions are also appropriately determined according to the type of cells used, etc. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is About 12 to about 72 hours.

  In step (b) of the above method, first, the expression level of the Polθ gene in the cell contacted with the test substance is measured. The expression level can be measured by a method known per se in consideration of the type of cells used. For example, when a cell capable of naturally expressing the Polθ gene is used as a cell capable of measuring the expression of the Polθ gene, the expression level is a method known per se for a product of the Polθ gene, such as a transcription product or a translation product. Can be measured. For example, the expression level of the transcript can be measured by preparing total RNA from cells and performing RT-PCR, Northern blotting, or the like. The expression level of the translation product can be measured by preparing an extract from the cells and using an immunological technique. As an immunological method, a radioisotope immunoassay (RIA method), an ELISA method (Methods in Enzymol. 70: 419-439 (1980)), a fluorescent antibody method, or the like can be used. On the other hand, when a cell capable of performing a reporter assay for the Polθ gene transcriptional regulatory region is used as a cell capable of measuring Polθ gene expression, the expression level can be measured based on the signal intensity of the reporter.

  Next, the expression level of the Polθ gene in the cells contacted with the test substance is compared with the expression level of the Polθ gene in the control cells not contacted with the test substance. The comparison of expression levels is preferably performed based on the presence or absence of a significant difference. The expression level of the Polθ gene in the control cells not contacted with the test substance is the expression level measured at the same time, even if the expression level was measured in advance compared to the measurement of the expression level of the Polθ gene in the cells contacted with the test substance. Although it may be present, it is preferably the expression level measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.

  In step (c) of the above method, a test substance that regulates the expression level of the Polθ gene is selected. The regulation of the expression level of the Polθ gene can be an increase or decrease in the expression level. For example, a test substance that increases the expression level (promotes the expression) of the Polθ gene can have an action of promoting humoral immune function, and can be a prophylactic / therapeutic agent for immunodeficiency diseases. On the other hand, a test substance that decreases the expression level of Polθ gene (suppresses expression) can have an action of suppressing humoral immune function and can be a prophylactic / therapeutic agent for autoimmune disease or allergic disease. Therefore, it becomes possible to select a drug or a candidate substance for a research reagent, such as a preventive / therapeutic agent for immune diseases, using the expression level of the Polθ gene as an index.

In another embodiment, the screening method of the present invention comprises the following steps (a) to (c):
(A) contacting the test substance with Polθ protein;
(B) a step of measuring the binding ability of the test substance to the Polθ protein;
(C) A step of selecting a test substance capable of binding to the Polθ protein based on the result of (b).

  In step (a) of the above method, the test substance is brought into contact with the Polθ protein. The contact of the test substance with the protein can be performed by mixing the test substance and the Polθ protein in a solution.

  Polθ protein can be prepared by a method known per se. For example, Polθ protein can be isolated and purified from the above-described tissue expressing Polθ gene. However, in order to prepare Polθ protein quickly, easily and in large quantities, and to prepare human Polθ protein, it is preferable to prepare a recombinant protein by a gene recombination technique. The recombinant protein may be prepared in either a cell system or a cell-free system.

  In step (b) of the above method, the ability of the test substance to bind to the Polθ protein is measured. The binding ability can be measured by a method known per se, for example, a binding assay, a method using surface plasmon resonance (for example, use of Biacore (registered trademark)). The binding ability of the test substance to the Polθ protein may also be measured in the presence of an antagonist to the Polθ protein (for example, a coupling factor of the Polθ protein). In this case, for example, the binding ability of the test substance to the Polθ protein can be measured using the change in the binding amount of the antagonist to the Polθ protein as an index.

  In step (c) of the above method, a test substance capable of binding to the Polθ protein is selected. A test substance capable of binding to the Polθ protein may be a substance that can regulate (for example, promote or suppress) the function of the Polθ gene. Therefore, this methodology can be useful, for example, as a first screening for substances that can regulate the function of the Polθ gene.

  The screening method of the present invention can also be performed by administering a test substance to an animal. Examples of the animal include mammals such as mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys, and the animals of the present invention. When the screening method of the present invention is performed using an animal, for example, a test substance that regulates the expression level of the Polθ gene can be selected.

  The screening method of the present invention enables screening for substances that can regulate immune function. Therefore, the screening method of the present invention is useful for the development of the above-mentioned pharmaceuticals or research reagents, or the above-mentioned regulator of B cell number.

(6. Method for identifying Polθ gene polymorphisms that cause changes in the ability to regulate immune functions)
The present invention also includes a method for identifying a Polθ gene polymorphism that causes a change in the ability to regulate immune function, comprising analyzing whether a specific polymorphism in the Polθ gene causes a change in ability to regulate immune function, Provided is a protein / nucleic acid molecule containing a Polθ gene polymorphism that causes a change in the ability to regulate immune function identified by the method.

  The polymorphism of the Polθ gene means a nucleotide sequence mutation found at a certain frequency in the genomic DNA containing the Polθ gene in a certain population, and substitution or deletion of one or more DNAs in the genomic DNA containing the Polθ gene. It can be additions (eg, SNPs, haplotypes), as well as repeats, inversions, translocations, etc. of partial regions in the genomic DNA. The polymorphism type of the Polθ gene identified by the method of the present invention is a nucleotide sequence that differs in frequency between animals suffering from immune diseases and animals not affected among all types of polymorphisms in the Polθ gene. For example, a change in the expression or function of the Polθ gene. In addition, although the animal used as the analysis object of Pol (theta) gene polymorphism is not specifically limited as above-mentioned, a human is preferable.

  The analysis step can be performed by a method known per se such as linkage analysis. When there is a significant difference in the frequency of possessing a specific genetic polymorphism depending on the presence or absence of an immune disease, subjects who have that type of genetic polymorphism have a relatively higher risk of developing the disease than subjects who do not Determined to be low.

  The identification method of the present invention may further include a step of subjecting a DNA sample prepared from a biological sample derived from a mammal to sequencing, and determining a new type of Polθ polymorphism. As a biological sample, not only a sample (for example, blood) containing Pol θ gene-expressing tissue or cells (for example, B cells) but also any tissue containing genomic DNA such as hair, nails, skin, and mucous membranes can be used. In view of availability, burden on the human body, and the like, the biological sample is preferably hair, nails, skin, mucous membrane, blood, plasma, serum, saliva and the like. Genetic polymorphism can be determined by analyzing a large number of nucleotide sequences of genomes or transcripts contained in biological samples of different origins and identifying mutations found at a certain frequency in the determined nucleotide sequences.

  The Polθ gene polymorphism that causes a change in the regulatory ability of the immune function determined by the identification method of the present invention and the method is useful for determining the risk of developing immune diseases.

(7. Determination method and diagnostic agent of immune disease)
(7.1. Determination method and diagnostic agent based on measurement of expression level)
The present invention provides a method for determining the onset or risk of developing an immune disease in an animal, comprising measuring the expression level of the Polθ gene using a biological sample of the animal.

In one embodiment, the determination method of the present invention includes the following steps (a) and (b):
(A) a step of measuring the expression level of the Polθ gene in a biological sample collected from an animal;
(B) A step of evaluating the onset or possibility of onset of an immune disease based on the expression level of the Polθ gene.

  In step (a) of the above method, the expression level of the Polθ gene is measured in a biological sample collected from an animal. The animal is not particularly limited as described above, but a human is preferable. The biological sample may be a sample (for example, blood) containing tissues or cells (for example, B cells) expressing Polθ gene, or cells separated from the sample. Further, the measurement of the expression level of the Polθ gene can be performed in the same manner as in the step (b) of the screening method of the present invention.

  In step (b) of the above method, based on the expression level of the Polθ gene, it is evaluated whether or not the animal suffers from an immune disease, or whether or not the animal is likely to suffer in the future. Specifically, first, the measured expression level of the Polθ gene is compared with the expression level of the Polθ gene in an animal that does not suffer from an immune disease (for example, a normal animal). The comparison of expression levels is preferably performed based on the presence or absence of a significant difference. The expression level of the Polθ gene in an animal not suffering from an immune disease can be determined by a method known per se.

  Next, from the comparison result of the expression level of the Polθ gene, it is determined whether or not the animal is likely to suffer from an immune disease, or whether or not the animal is likely to be affected in the future. It is known that in animals that develop a specific disease, changes in the expression of genes associated with the disease are often observed. It is also known that changes in the expression of specific genes are often observed before the onset of specific diseases. In fact, the animal of the present invention, which can be an immunodeficiency disease model, has a decreased expression of the Polθ gene. Further, from the findings disclosed in the present specification, when the expression of the Polθ gene is increased, the possibility of suffering from an autoimmune disease or allergic disease or the possibility of suffering in the future is relatively It is thought that it is expensive. Therefore, it is possible to determine the onset or risk of developing an immune disease by analyzing the expression level of the Polθ gene.

  The present invention also provides a diagnostic agent for the onset or risk of developing an immune disease, comprising a reagent for measuring the expression level of the Polθ gene.

The reagent for measuring the expression level of the Polθ gene is not particularly limited as long as the expression of the Polθ gene can be quantified. For example, an antibody against the Polθ protein (described above), a nucleic acid probe against the Polθ gene transcription product, or a Polθ gene transcription product can be used. It may contain a plurality of amplifiable primers. These may or may not be labeled with a labeling substance. When not labeled with a labeling substance, the diagnostic agent of the present invention can further contain the labeling substance. Examples of the labeling substance include fluorescent substances such as FITC and FAM, luminescent substances such as luminol, luciferin, and lucigenin, radioisotopes such as ³ H, ¹⁴ C, ³² P, ³⁵ S, and ¹²³ I, biotin, and streptavidin. And the like, and the like.

  The nucleic acid probe for the Polθ gene transcription product may be either DNA or RNA, but DNA is preferred in consideration of stability and the like. The probe may be either single-stranded or double-stranded. The size of the probe is not particularly limited as long as the transcript of the Polθ gene can be detected, but is preferably about 15 to 1000 bp, more preferably about 50 to 500 bp. The probe may be provided in a form fixed on a substrate like a microarray.

  A plurality of primers (eg, primer pairs) capable of amplifying the Polθ gene are selected such that a detectable size nucleotide fragment is amplified. A detectable size nucleotide fragment can have a length of, for example, about 100 bp or more, preferably about 200 bp or more, more preferably about 400 bp or more. The size of the primer is not particularly limited as long as the Polθ gene can be amplified, but may preferably be about 15 to 100 bp, more preferably about 18 to 50 bp, and still more preferably about 20 to 30 bp. When the means capable of quantifying the Polθ gene transcript is a primer, the diagnostic agent of the present invention can further contain a reverse transcriptase.

  The determination method and diagnostic agent of the present invention enable determination of the presence or absence of an immune disease or the possibility of suffering from the disease. Therefore, the present invention is useful, for example, for easy and early detection of immune diseases.

(7.2. Determination method and diagnostic agent based on measurement of polymorphism)
The present invention provides a method for determining the risk of developing an immune disease in an animal, comprising measuring a polymorphism of the Polθ gene using a biological sample of the animal.

In one embodiment, the determination method of the present invention includes the following steps (a) and (b):
(A) a step of measuring a Polθ gene polymorphism in a biological sample collected from an animal;
(B) The process of evaluating the onset possibility of an immune disease based on the type of polymorphism.

  In step (a) of the above method, a polymorphic type of Polθ gene is measured in a biological sample collected from an animal. The animals are as described above. The biological sample can be the same as described above in the identification method of the present invention.

  The measurement of the polymorphic type can be performed by a method known per se. For example, RFLP (restriction fragment length polymorphism) method, PCR-SSCP (single-stranded DNA higher-order structure polymorphism analysis) method, ASO (Allele Specific Oligonucleotide) hybridization method, direct sequencing method, ARMS (Amplification Refracting Mutation) System) method, denaturing gradient gel electrophoresis (Denaturing Gradient Gel Electrophoresis) method, RNase A cleavage method, DOL (Dye-labeled Oligonucleotide Ligation) method, TaqMan PCR method, invader method, etc. can be used.

  In step (b) of the above method, based on the type of polymorphism, it is evaluated whether the animal is likely to suffer from an immune disease or not. It is known that animals that are likely to develop a particular disease often have a particular type of polymorphism in a gene associated with that disease. In fact, the animal of the present invention, which can be an immunodeficiency disease model, has a decreased expression of the Polθ gene. Therefore, it is considered that an animal containing a polymorphism that reduces the function of the Polθ gene has a relatively high possibility of developing an immunodeficiency disease. Similarly, an animal containing a polymorphism that enhances the function of the Polθ gene is considered to have a relatively high possibility of developing an autoimmune disease or an allergic disease. Therefore, it is possible to determine the possibility of developing an immune disease by analyzing the polymorphism. In addition, the type of polymorphism to be measured in this step can be obtained by, for example, the identification method of the present invention.

  The present invention also provides a diagnostic agent for the risk of developing an immune disease, which comprises a reagent for measuring a polymorphism of the Polθ gene.

  The reagent for measuring the polymorphism of the Polθ gene is not particularly limited as long as the polymorphism of the Polθ gene can be determined. The means may be labeled with a labeling substance. Further, when the means is not labeled with a labeling substance, the kit can further include the labeling substance.

  Specifically, the reagent for measuring the polymorphism of the Polθ gene is a nucleic acid probe capable of specifically measuring the Polθ gene having a specific type of polymorphism, or specifically the Polθ gene having a specific type of polymorphism. It may contain a plurality of primers that can be amplified. The nucleic acid probe and primer can be for genomic DNA containing Polθ gene or Polθ gene transcript. The nucleic acid probe and primer may be provided together with a transcription product or a reagent for extracting genomic DNA.

  The nucleic acid probe capable of specifically measuring a Polθ gene having a specific type of polymorphism is not particularly limited as long as it can select a Polθ gene having a specific type of polymorphism. The probe may be either DNA or RNA, but DNA is preferable in consideration of stability and the like. The probe may be either single-stranded or double-stranded. The size of the probe is preferably as short as possible so that a Polθ gene having a specific type of polymorphism can be selected. For example, the size of the probe may be about 15 to 30 bp. The probe may be provided in a form fixed on a substrate like a microarray. The probe enables, for example, an ASO (Allele Specific Oligonucleotide) hybridization method.

  A plurality of primers (eg, primer pairs) capable of specifically amplifying a Polθ gene having a particular type of polymorphism are selected such that a measurable size nucleotide fragment is amplified. Such a plurality of primers is designed to include a polymorphic site at the 3 'end of any one of the primers, for example. Nucleotide fragments of measurable size can have a length of, for example, about 100 bp or more, preferably about 200 bp or more, more preferably about 400 bp or more. The size of the primer is not particularly limited as long as the Polθ gene can be amplified, but may preferably be about 15 to 100 bp, more preferably about 18 to 50 bp, and still more preferably about 20 to 30 bp. When the means capable of measuring the polymorphism of the Polθ gene is a primer pair for the Polθ gene transcript, the determination kit can further include a reverse transcriptase.

  Examples of the reagent for measuring a polymorphism of the Polθ gene include those containing a restriction enzyme that recognizes a specific type of polymorphic site. According to such a reagent, polymorphism analysis by RFLP becomes possible.

  The determination method and diagnostic agent of the present invention enable determination of the risk of developing an immune disease. Therefore, the determination method and diagnostic agent of the present invention are useful because they provide an opportunity for improving lifestyle habits aimed at preventing immune diseases.

  The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Example 1: Production of Polθ-deficient mice
1.1. Construction of targeting vector The targeting vector was constructed as shown in FIG. First, a mouse EST clone (Genbank accession number: B1656994) showing homology to a partial sequence of the human Polθ gene (Sharief et al., Genomics 59: 90-96, 1999) was obtained by homology search. A cDNA fragment corresponding to this EST clone was cloned from mouse germinal center B cells by PCR. This fragment was labeled in the presence of ³² P-dCTP by the random prime label method. The labeled fragment was used as a probe to screen a 129SVJ mouse genomic library. As a result, when the restriction enzyme cleavage site and the base sequence of the positive phage clone were examined, phage clones (Polθ1-2, Polθ20-1) having two independent independent genomic DNA fragments of Polθ gene were obtained. The base sequence of cDNA encoding mouse Polθ protein is known and disclosed, for example, in GenBank Accession No .: AY074936. Polθ1-2 was a fragment of about 13.3 kb in total length including exon 25-27, and Polθ20-1 was a fragment of about 15 kb in total length including exon 26-28.
Next, DNA was extracted from the phage clones Polθ1-2 and Polθ20-1, and cut with NotI, followed by 0.7% agarose gel electrophoresis, and Polθ1-2 (about 13.3 kb) and Pol θ20-1 (about 15 kb). ), And ligated with plasmid pBluescript II KS + (Stratagene) cleaved with NotI using T4 ligase, transformed into E. coli DH5α, and plasmids (pBS-Polθ1-2 and pBS-Polθ20-1) Got. pBS-Polθ1-2 was cleaved with PvuII and subjected to 1% agarose gel electrophoresis. A DNA fragment corresponding to about 3.8 kb was recovered and used as a 5 ′ homology region. Similarly, pBS-Polθ20-1 was cleaved with SphI, and a fragment of about 2.9 kb was recovered, which was used as the 3 ′ homology region. Finally, a targeting vector was prepared by introducing a 5 ′ homology region, a neomycin resistance gene, and a 3 ′ homology region into pBluescript SK− in this order.

1.2. Preparation of Polθ-deficient ES cells The targeting vector was cleaved with PvuI to obtain linear DNA (1 mg / ml). A linear targeting vector (30 μg / 30 μl) was transfected into mouse ES cells (5 × 10 ⁷ cells / 400 μl) by electroporation (260 V, 500 μF, room temperature), and G418 (400 μg / ml) was added after 2 days of culture. The culture was carried out for 7 days in the medium. DNA was extracted from a part of the resulting ES cell colonies, and clones in which only homologous recombination occurred were identified by the PCR method. In addition, when Southern blot analysis was performed using the genomic DNA of the 3 ′ portion not included in the targeting vector as a probe, a 4.4 Kb DNA fragment was detected by homologous recombination with 7.7 Kb of the wild type. I was able to. From the above, it was confirmed that Polθ-deficient ES cells were obtained.

1.3. Production of Polθ-deficient mice from Polθ-deficient ES cells Female mice were treated with 5 marginal gonadotropins (pregnant mare's serum gonadotropin, PMSG: imperial organ, Tokyo) and human chorionic gonadotropin (human). Chorionic gonadotropin, hCG: Gonatropin: Imperial organ, Tokyo) 2.5 international units were each administered intraperitoneally, and 8-cell embryos were obtained by the fallopian tube-uterine reflux method on day 2.5 of fertilization. A small depression was made in the culture dish, and the resulting 8-cell embryo and ES cell mass (including 8-20 cells) were mixed and cultured in the depression for 27 hours. A pseudopregnant mouse was prepared by mating a vas deferens male mouse and a normal female mouse, and the cells after the mixed culture were transplanted into the egg collection tube of the pseudopregnant mouse. Pseudopregnant mice were anesthetized with 50 mg / kg body weight pentobarbital sodium (Nembutal: Abbott Laboratories), incised about 1 cm in both tendons to expose the ovaries and fallopian tubes, and tweezers of the ovarian sac under a stereomicroscope An incision was made to expose the fallopian tube, and then about 20 manipulated eggs per oviduct were fed into the fallopian tube. Thereafter, the fallopian tube and ovary were returned to the abdominal cavity and both incisions were sutured. A 100% chimeric mouse having a body color of agouti was born from a mouse that became pregnant after transplanting the manipulated egg. The resulting 100% chimeric mouse was crossed with C57BL / 6 to obtain heterozygous mice, and Polθ KO mice were obtained by crossing heterozygous mice.
The resulting mouse genotype was then confirmed by the size of the DNA fragment generated by PCR. First, the tail of the mouse was cut about 5 mm and digested with proteinase K (0.25 mg / ml) (55 ° C., overnight). Genomic DNA was extracted according to a conventional method and dissolved in 100-200 μl of distilled water to obtain a PCR template. A sequence contained in the Neo gene (Neo / s: 5'-TCGCCTTCTATCGCCTTCTT-3 ': SEQ ID NO: 2) and Neo / as: 5'-ATAGCCCGAATAGCTCTCTCCA-3': SEQ ID NO: 3) and two sites of the Polθ gene (s2 : 5′-GGGTTGTTTGTAGGCACTGA-3 ′: SEQ ID NO: 4) and (as2: 5′-ATCGGCAGGGTAGCATCATCAT-3 ′: SEQ ID NO: 5) were designed and PCR was performed. Mutated genes yield 4.2 kb and 4.1 kb PCR products, respectively. In fact, it was confirmed that a PCR product as expected was generated and Polθ KO mice were obtained.

Example 2: Purification of Polθ-deficient splenic B cells from Polθ-deficient mice B cells were purified using a commercially available B cell enrichment kit (Becton Dickinson, catalog number: 557772). First, spleen cells were collected from Polθ-deficient mouse spleen and reacted with biotinylated antibodies (anti-CD4 antibody, anti-CD43 antibody and anti-TER119 antibody) that recognize cells other than B cells. Polθ-deficient spleen B cells were purified by removing cells bound to the biotinylated antibody using avidin magnetic beads, and then recovering antibody-unbound cells remaining in the test tube. The purity of Polθ-deficient splenic B cells was 95% or more.

Reference Example 1: In the bone marrow of Polθ-deficient mice, cells were collected from the bone marrow of 12-20 weeks old Polθ-deficient mice in which B cells decreased . The collected cells are subjected to propidium iodide staining to calculate the proportion of living cells (PI ^- cells) in bone marrow cells, and stained with antibodies against cell surface markers (B220, CD43, IgM) of each B cell. Then, by subjecting to FACS, the ratio of each B cell in the bone marrow cells was calculated. The ratio was calculated for each of the six mice.
As a result, B cells, particularly pre-B cells, immature B cells, and mature B cells were decreased in the bone marrow of all analyzed Polθ-deficient mice (FIG. 2). The total number of B cells in the bone marrow serves as an index indicating the presence or absence of abnormal B cell differentiation / proliferation. For example, a decrease in the number of B cells usually indicates a decrease in the proliferation ability of B cells in the bone marrow or abnormal differentiation.
From the above, it was suggested that Polθ-deficient mice can be model mice for immune diseases caused by decreased B cell proliferation ability or abnormal differentiation.

Reference Example 2: Pol θ-deficient B cells include proliferation responsiveness to antigen as an important index for measuring the properties of B cells whose proliferation responsiveness to antigen stimulation specifically decreases . Usually, if the responsiveness to an antigen or a stimulus that mimics the antigen (for example, a stimulus by an anti-IgM antibody) is enhanced, it means that the B cell is easily activated. On the other hand, if the responsiveness to antigen stimulation decreases, it means that it is difficult to activate. Therefore, based on the result of Reference Example 1, in order to analyze B cells of Polθ-deficient mice in more detail, the proliferation responsiveness of Polθ-deficient B cells to antigen stimulation was examined.
First, spleen B cells were purified from Polθ-deficient mice according to the method described in Example 2. The purified cells were then cultured for 48 hours in the presence of anti-IgM antibody (Fab ₂ ) (2 μg / ml and 0-30 μg / ml doses). [ ³ H] -labeled thymidine was added to the culture solution 6 hours before the end of the culture, and the proliferation responsiveness of Polθ-deficient B cells was evaluated based on the [ ³ H] count incorporated into the cultured cells. As controls for anti-IgM antibodies, CD40L (factor having B cell proliferation inducing ability: 2 μg / ml), CD40L (2 μg / ml) + anti-CD8 antibody (anti-CD8 antibody enhances CD40L stimulation: 2 μg / ml) ), IL4 (0.02 μg / ml) was used. In addition, normal B cells purified from wild-type mice were subjected to the same treatment, and the proliferation responsiveness of Polθ-deficient B cells and normal B cells were compared.
As a result, Polθ-deficient B cells had a specific decrease in proliferation responsiveness to anti-IgM antibodies as compared to normal B cells (FIGS. 3A and 3B). This indicates that Polθ-deficient mice react weakly to foreign antigens and can cause diseases caused by attenuated immune responses (eg, immunodeficiency diseases).
From the above, it was shown that Polθ-deficient B animals serve as animal models of diseases caused by attenuated immune responses.

Reference Example 3: Polθ-deficient B cells are promoted by antigen stimulation to induce apoptosis and decrease in mitotic cells. The decrease in proliferation response of Polθ-deficient B cells to antigen stimulation confirmed in Reference Example 2 is the cell cycle. It was thought that this could be due to inhibition of the progression of. Thus, the state of the cell cycle and the proportion of apoptotic cells were examined for Polθ-deficient B cells stimulated with antigen.
First, as in Reference Example 2, Polθ-deficient B cells were cultured for 48 hours. The cultured cells were treated with a surfactant, and then RNA was degraded with RNase. Subsequently, intracellular DNA content was measured by dyeing intracellular DNA with propidium iodide. Normally, the amount of DNA in stationary cells is 2N, and the DNA content increases to 4N (2 times) at the mitotic phase. In addition, since the DNA content of cells in apoptosis decreases to 2N or less, the state of the cell cycle and the proportion of apoptotic cells can be analyzed by this assay. Normal B cells purified from wild type mice were also subjected to the same treatment, and the proliferation responsiveness of Polθ-deficient B cells and normal B cells were compared.
As a result, in Polθ-deficient B cells stimulated with anti-IgM antibody (2 μg / ml), the ratio of apoptosis increased and cells in mitotic phase (S and G2 / M) decreased compared to normal B cells (FIG. 4). (A)). On the other hand, in the control (CD40L stimulation), there was no difference in apoptosis and cell cycle progression (FIG. 4B).
From the above, it was shown that the specific decrease in the proliferation responsiveness of Polθ-deficient B cells to antigen stimulation can be attributed to the promotion of apoptosis and the reduction of cells in the mitotic phase.

  The animal and cell of the present invention can be used as a model animal and cell of an immunodeficiency disease, and enables analysis of Polθ gene and immune function regulation mechanism. The targeting vector of the present invention enables the production of the animals and animal cells of the present invention. The composition for regulating immune function of the present invention can be used as a medicine for an immune disease such as an immunodeficiency disease, an autoimmune disease, an allergic disease, a research reagent and the like. The screening method of the present invention enables the development of pharmaceuticals and research reagents for immune diseases. The determination method and diagnostic agent of the present invention make it possible to evaluate the onset or risk of developing immune diseases.

It is a figure which shows the strategy of targeting vector construction. Abbreviations are as follows: Neo: Neomycin Neo / s: Primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 Neo / as: Primer consisting of the nucleotide sequence shown in SEQ ID NO: 3: Shown in SEQ ID NO: 4 Primer comprising the nucleotide sequence as2: Primer comprising the nucleotide sequence represented by SEQ ID NO: 5 It is a figure which shows the ratio of the various cells (PI ^- cell, B220 ⁺ cell, pro B cell, pre-B cell, immature B cell, mature B cell) in the bone marrow of a Pol (theta) deficient mouse. The measurement was performed in 6 independent trials, and the average was calculated. The result of each trial is indicated by six symbols, and the average is indicated by a horizontal line. It is a figure which shows the proliferation responsiveness of Pol (theta) deficient B cell with respect to antigen stimulation. (A) is a figure which shows the response of Pol (theta) deficient B cell with respect to various antigens (anti-IgM antibody, CD40L, anti-CD8 antibody, IL4). (B) is a graph showing the dose response of Polθ-deficient B cells to anti-IgM antibody stimulation. It is a figure which shows the ratio of each phase of a cell cycle after anti- IgM antibody and CD40L stimulation in Pol (theta) deficient B cell.

Claims (40)

A non-human animal containing a functional defect of the Polθ gene. The animal according to claim 1, which is a transgenic animal. The animal according to claim 1, wherein the number of B cells is reduced. The animal according to claim 1, which is an immunodeficiency disease model. The animal according to claim 4, wherein the immunodeficiency disease is a disease accompanied by suppression of humoral immune function. A method for producing a non-human chimeric animal containing a functional defect of the Polθ gene, comprising the following steps (a) to (c):
(A) providing an embryonic stem cell containing a functional defect of the Polθ gene;
(B) introducing the embryonic stem cell into an embryo to obtain a chimeric embryo;
(C) Transplanting the chimeric embryo into an animal to obtain a chimeric animal. A method for producing a non-human transgenic animal containing a functional defect of the Polθ gene, comprising the following steps (a) to (d):
(A) providing an embryonic stem cell containing a functional defect of the Polθ gene;
(B) introducing the embryonic stem cell into an embryo to obtain a chimeric embryo;
(C) transferring the chimeric embryo to an animal to obtain a chimeric animal;
(D) A step of mating the chimeric animal to obtain a Polθ gene-deficient heterozygote. The method according to claim 7, further comprising the step of mating Polθ gene-deficient heterozygotes to obtain Polθ gene-deficient homozygotes. Animal cells containing a functional defect of the Polθ gene. The animal cell according to claim 9, which is a transgenic cell. The animal cell according to claim 10, which is a cell derived from a non-human transgenic animal containing a functional defect of the Polθ gene. The animal cell according to claim 9, which is an embryonic stem cell. The animal cell according to claim 9, which is a B cell. A method for producing an animal cell containing a functional defect of the Polθ gene, comprising the following steps (a) to (c):
(A) providing a targeting vector capable of inducing homologous recombination of Polθ gene;
(B) introducing the targeting vector into animal cells;
(C) A step of selecting cells that have undergone homologous recombination from animal cells into which the targeting vector has been introduced. A method for producing an animal cell comprising a functional defect of the Polθ gene, comprising the step of isolating cells from a non-human transgenic animal comprising a functional defect of the Polθ gene. A targeting vector capable of inducing homologous recombination of a Polθ gene, comprising a first polynucleotide and a second polynucleotide homologous to the Polθ gene, and a selection marker. A method for producing a targeting vector capable of inducing homologous recombination of a Polθ gene, comprising inserting a first polynucleotide and a second polynucleotide homologous to the Polθ gene, and a selection marker into the vector. A composition for regulating immune function, comprising a substance that regulates the expression or function of Polθ gene. The composition according to claim 18, which is a regulator of B cell number. The composition according to claim 18, comprising a substance that promotes the expression or function of the Polθ gene. 21. The composition according to claim 20, wherein the substance that promotes the expression or function of the Polθ gene is an expression vector comprising a Polθ protein or a nucleic acid encoding the Polθ protein. The composition according to claim 18, comprising a substance that suppresses the expression or function of the Polθ gene. The composition according to claim 22, wherein the substance that suppresses the expression or function of the Polθ gene is any one of (i) and (ii) below:
(I) antisense nucleic acid, ribozyme or RNAi-inducible nucleic acid molecule, or an expression vector comprising these;
(Ii) An expression vector comprising an antibody or a dominant negative mutant, or a nucleic acid encoding them. 19. A composition according to claim 18 which is a medicament. The composition according to claim 24, which is a prophylactic / therapeutic agent for immunodeficiency diseases. 26. The composition according to claim 25, wherein the immunodeficiency disease is a disease involving suppression of humoral immune function. A screening method for a substance capable of regulating an immune function, comprising evaluating whether a test substance can regulate the expression or function of a Polθ gene. 28. The method according to claim 27, wherein the substance capable of regulating immune function is a substance capable of regulating the number of B cells. 28. The method of claim 27, comprising assessing whether the test substance can promote Polθ gene expression or function. 28. The method according to claim 27, comprising evaluating whether the test substance can suppress the expression or function of the Polθ gene. 28. The method of claim 27, comprising the following steps (a) to (c):
(A) contacting the test substance with a cell capable of measuring the expression of the Polθ gene;
(B) measuring the expression level of the Polθ gene in a cell contacted with the test substance, and comparing the expression level with the expression level of the Polθ gene in a control cell not contacted with the test substance;
(C) A step of selecting a test substance that regulates the expression level of the Polθ gene based on the comparison result of (b). 28. The method of claim 27, comprising the following steps (a) to (c):
(A) contacting the test substance with Polθ protein;
(B) a step of measuring the binding ability of the test substance to the Polθ protein;
(C) A step of selecting a test substance capable of binding to the Polθ protein based on the result of (b). 28. The method according to claim 27, which is a method for screening a medicine. The method according to claim 33, which is a screening method for a prophylactic / therapeutic agent for an immunodeficiency disease. 35. The method of claim 34, wherein the immunodeficiency disease is a disease involving suppression of humoral immune function. A method for identifying a Polθ gene polymorphism that causes a change in the ability to regulate immune function, comprising analyzing whether a specific polymorphism in the Polθ gene causes a change in ability to regulate immune function. A method for determining the onset or risk of developing an immune disease in an animal, comprising measuring the expression level of the Polθ gene using a biological sample of the animal. A diagnostic agent for the onset or risk of developing immune diseases, comprising a reagent for measuring the expression level of the Polθ gene. A method for determining the risk of developing an immune disease in an animal, comprising measuring a polymorphism of the Polθ gene using a biological sample of the animal. A diagnostic agent for the risk of developing an immune disease, comprising a reagent for measuring a polymorphism of Polθ gene.

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