JP2007054073A - Nkt cell deficient animal and nkt cell animal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an experimental animal for clarifying the function and differentiation mechanism of NKT cell which is a lymphocyte subset having two kinds of receptors consisting of T cell receptor and NK receptor, clarifying the disease occurred in connection with the action of NKT cell and the research for the treating method, etc., of the disease. <P>SOLUTION: The invention provides an NKT cell deficient mouse in which the deficiency of NKT cell is caused by the deficiency of uniform Vα14Jα281α chain or Jα281 gene and a non-human animal containing only Vα14 positive NKT cell as non-mature lymphocyte. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an NKT cell-deficient non-human animal and a non-human animal having only Vα14-positive NKT cells as mature lymphocytes (hereinafter referred to as “NKT cell non-human animal”). The animal of the present invention is extremely useful as an experimental animal not only for clarifying the function and differentiation mechanism of NKT cells, but also for elucidating diseases caused by the action of NKT cells and for researching their treatment methods. is there.

NK1.1 antigen has been conventionally considered to be a specific marker for natural killer cells (hereinafter referred to as “NK cells”), but cells that also express NK1.1 antigen among T cells, That is, it has become clear that NK1.1 positive αβ T cells (hereinafter referred to as “NKT cells”) exist. The NKT cells are a unique lymphocyte subset with two antigen receptors, the T cell receptor (TCR) and the NK receptor. NKT cells express repertoire different from normal T cells and NK cells. For example, with respect to the β chain, 90% or more of NKT cells mainly express a limited repertoire of Vβ8.2 and other Vβ7 and Vβ2, and the α chain mainly expresses a uniform Vα14Jα281. There are also NKT cells that do not express Vα14 on the cell surface.
Hereinafter, in the present specification, the present invention is described only for Vα14-positive NKT cells that express Vα14TCR, and unless otherwise specified, NKT cells refer to Vα14-positive NKT cells.
Vα14-positive NKT cells differentiate and proliferate in extrathymic tissues and express a uniform TCRα chain (Vα14) and a limited TCRβ chain (mainly Vβ8.2). This uniform TCRα chain is created as a result of the Vα14 gene and the Jα281 gene being selected from the V and J gene groups upon reconstitution of the TCR gene and reconstitution on the genome.
(Yoshimoto T and Paul. WE CD4pos, NK1.1 posT cells promptly production interleukin 4 in response to in vivo challenge with anti-CD3, J Exp. Arase H, Arase N and Saito T. Interferonγ production by natural killer (NK) cells and NK1.1 + Tcells up NKR-P1 crosslinking. J Exp Med 183: 2391, 1996

NKT cells have Fas-Fas ligand-dependent and perforin-dependent cytotoxicity and various lymphokines (for example, IL-4, γ-IFN, IL-2) production ability. It is thought to be involved in the reaction. In particular, NKT cells have been reported to exhibit antitumor cytotoxic activity that seems to be mediated by perforin, and to produce IL-4 and IFN-γ very strongly against TCR stimulation (Yoshimoto T and Paul.W.E.CD4 ^pos , NK1.1 ^pos T cells promptly production interleukin 4 in response to in vivo challenge and CDN. ^{by natural killer (NK) cells and} NK1.1 + Tcells upon NKR-P1 crosslinking.J xp Med 183: 2391,1996). Furthermore, it is considered that a decrease in Vα14-positive NKT cells has a strong correlation with the onset of autoimmune diseases. Therefore, elucidating the function of NKT cells and the mechanism of differentiation is expected to greatly contribute to the elucidation of the pathogenesis of autoimmune diseases and allergic diseases, transplanted bone marrow rejection, and tumor immunity.
However, in conventional NKT cell research, there is no NKT cell line, and hybridoma cells produced by Bendelac et al. Cannot confirm the expression of Vα14TCR. Therefore, NKT cell functions other than using NKT cells isolated from individuals. It was a situation that could not be analyzed. However, since the number of NKT cells in the peripheral lymphoid tissues of animals is small, development of a system rich in NKT cells has been strongly demanded.
It is desirable to examine the functions of NKT cells involved in various immune responses in vivo. In vivo, lymphocytes other than NKT cells, such as T cells, B cells, and NK cells, act in a complex manner, so that the functions of NKT cells are separated from other lymphocyte factors. In order to analyze clearly, animals that produce other mature lymphocytes such as T cells, B cells, NK cells, but not NKT cells (NKT cell-deficient animals), T cells, B cells, NK An animal (NKT cell animal) that produces only NKT cells without producing other mature lymphocytes such as cells has been strongly desired. However, at present, such genetically stable and systematic mutant animals are not known.

In order to solve the above-mentioned problems, the present inventor has conducted extensive research, and as a result, the Vα14 gene and the Vβ8.2 gene are involved in the determination of differentiation from lymphocyte progenitor cells to Vα14-positive NKT cells. And that Vα14TCR is required for differentiation of NKT cells, and that Vβ8.2 TCR is involved in the determination of preferential differentiation from common progenitor cells of NK cells and NKT cells to NKT cells, The inventors have invented the NKT cell-deficient animal and NKT cell animal of the present invention.
The present invention relates to a non-human animal deficient in NKT cells, a non-human animal having only Vα14-positive NKT cells as mature lymphocytes (a non-human animal having immunodeficiency, in which Vα14 and Vβ8.2 are expressed). An animal characterized by being an animal).

In the present invention, “an animal deficient in NKT cells” or “an animal deficient in NKT cells” refers to an animal that does not produce NKT cells. It is preferable that the defect of the NKT cell is caused by the defect of the Vα14Jα281α chain.
In the present invention, the “Vα14Jα281α chain” refers to a TCRα chain expressed by the rearranged TCR gene Vα14Jα281.
The uniform deletion of the Vα14Jα281α chain is caused by deletion of at least one of the Vα14 gene and the Jα281 gene constituting the reconstituted TCR gene. Two Vα14 genes are present apart from each other on the genomic DNA, whereas only one Jα281 gene is present on the genomic DNA, so that deletion of the Jα281 gene can be caused more easily. It is preferred to delete the copy of the Jα281 gene.
In order to delete the Jα281 gene, a deletion may be caused in a part of the Jα281 gene, a point mutation may be introduced, or another gene may be inserted.
As another gene to be inserted, a marker gene used for positive selection of cells into which the gene has been inserted, such as a neomycin (neo) resistance gene, is preferable. This neomycin resistance gene enables selection of a target gene by using G418, which is a neomycin analog. In addition, a marker gene used for negative selection in order to select and remove a target gene can be used together with the positive selection marker gene.
Although the insertion location of the marker gene used for the purpose of positive selection along with the functional defect of the gene is not particularly limited, it is usually inserted so that the exon is defective.

As used herein, “an animal having only Vα14-positive NKT cells as mature lymphocytes” refers to an animal having only Bα14-positive NKT cells as mature lymphocytes without B cells, T cells and NK cells. . Such an animal should be prepared as an animal having an immunodeficiency, for example, a transgenic animal in which a transgenic Vβ8.2 gene and a transgenic Vα14 gene are expressed in a RAG-1 deficient, RAG-2 deficient, or SCID animal. Can do.
In the present specification, “Vα14tg” represents that a transgenic Vα14 gene was expressed, and “Vβ8.2tg” represents that a transgenic Vβ8.2 gene was expressed.
“Immunodeficiency” as used herein refers to a phenotype that lacks all or only B cells and T cells. For example, taking mice as examples, SCID mice, RAG-1 deficient (RAG-1 ^{− / −} ) mice, RAG-2 deficient (RAG-2 ^{− / −} ) mice, TCRα deficient (TCRα ^{− / −} ), etc. Can be mentioned.
SCID mice are severe combined immunodeficiency mice that lack mature T cells and B cells. This mouse is caused by a deletion of the SCID gene involved in VDJ rearrangement of the TCR gene and immunoglobulin gene (B cell receptor gene) present on chromosome 16.
RAG-1 ^{− / −} mice and RAG-2 ^{− / −} mice are mice that lack mature B cells and T cells. These mice are caused by the deletion of the RAG-1 and RAG-2 genes that induce V (D) J rearrangement of the T cell receptor (TCR) and B cell receptor genes present on mouse chromosome 2.
TCRα ^{− / −} mice are immunodeficient mice that lack only mature T cells. This mouse is caused by a defect in the TCR α chain constant region gene present on the mouse 14 chromosome.
The TCR gene encodes a specific TCR protein by gene rearrangement. At this time, a phenomenon called allelic exclusion usually occurs. For the TCR β chain gene, if a transgenic Vβ 8.2 gene is present, it is a gene that has already been reconstituted before the endogenous TCR undergoes gene reconstitution, so allelic exclusion works and endogenous TCR β chain gene rearrangement is suppressed. However, regarding the TCRα chain gene, even if the transgenic Vα14 gene is present, the allele exclusion is incomplete, so that the endogenous TCRα chain gene is rearranged and expressed. In order to eliminate the influence of such an endogenous TCR α chain gene, a transgenic body of an animal having an immunodeficiency as described above is prepared.
The term “non-human animal” as used in the present invention refers to all animals except humans, preferably mammals, more preferably rodents, and more preferably mice.
Hereinafter, the present invention will be described using a mouse as an example, but the present invention is not limited to this example.

Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1 Creation of NKT cell mouse (A) Elucidation of genes necessary for differentiation of NKT cells NKT cells express a uniform TCR α chain (Vα14Jα281) and a limited β chain (mainly Vβ8.2). Thus, the role of these TCRs in the differentiation of NKT cells was examined using various TCR transgenic (tg) mice.
(Method) TCRαtg (Vα14, Vα1, Vα11) mice and these mice and TCRα-deficient (TCRα ^{− / −} mice (Mobaerts, P., Clarke, AR, Rudnicki, MA, Iacomini, J., Itohara, S., Lafile, JJ, Wang, L., Ichikawa, Y., Jaenisch, R., Hopper, ML & Tonegawa, S. (1992) Nature (London) 360, 225-231. ) Expression of NKT cells in mice (TCRα ^{− / −} / Vα14, TCRα ^{− / −} / Vα1), TCRβtg (Vβ8.2, Vβ3) mice and TCRαβtg (Vα14 / Vβ8.2, Vα11 / Vβ3) mice FACS and RNase It was measured by Tekushon analysis.
The Vα14tg mouse was prepared by the following method.
The T cell receptor Vα14Jα281Cα cDNA (pTsα104 / 46) 1.0 kb cloned from the KLH-specific suppressor T cell hybridoma 34S-281 cDNA library was replaced with a 4.3 kb human β-actin promoter (Leavitt, J., Gunning). , P., Porreca, P., Ng, S.-Y., Lin, C.-S. & Kedes, L. (1984) Mol. Cell. Biol., 1961-1969). The expression vector pHβAPr-Tsα2-neo was prepared by introducing it into the SalI restriction enzyme site of neo (FIG. 1). The 11.0 kb BglII / PvuI fragment obtained from this pHβAPr-Tsα2-neo was microinjected into fertilized eggs derived from C57BL / 6 mice and returned to the foster tube of the foster parent. DNA was extracted from the tail of the born mouse, the integration of the Vα14TCR gene was confirmed by Southern plot analysis, and a Vα14TCR gene transgenic mouse (Vα14tg mouse) was prepared.
(result)
NKT cells appeared only for Vα14tg, Vβ8.2tg, and Vα14 / Vβ8.2tg, but not for other tg.
In Vβ8.2tg, NK cells disappeared and all differentiated into NK1.1 ⁺ Vβ8.2T cells, indicating the importance of Vβ8.2TCR in determining differentiation from common progenitor cells to NK cells and NKT cells. It became clear.
In TCRα ^{− / −} / Vα14tg, normal T cells did not appear and all became NK1.1 ⁺ Vα14 ⁺ T cells, confirming the necessity of Vα14 TCR in differentiation of NKT cells.
From the above, it was considered that a combination of Vα14 gene and Vβ8.2 gene is necessary to differentiate only NKT cells.

(B)
(1) Vα14tg mice obtained according to the above method (A) were RAG-1 gene knockout mice (RAG-1 ^{− / −} mice) (provided by Dr. Mombaets, Monbaets P, Iacomini J, Johnson RS, et al. RAG-1 delicate, rice have nomature B and T-lymphocytos, Cell 68: 869, 1992). FIG. 2 shows a technique for deleting the RAG-1 gene in order to generate RAG-1 ^{− / −} mice. First, a knockout construct in which the RAG-1 gene was disrupted as shown in FIG. 2 (1) was prepared by inserting the neo gene into the RAG-1 gene. This knockout construct was injected into ES cells derived from a 129SV mouse having a RAG-1 gene-containing genome represented by the restriction enzyme map of FIG. 2 (2), and between the knockout construct and the 129SV mouse genome, FIG. Preparative recombination was caused at the X site in (1) and (2). As a result of this homologous recombination, as shown in FIG. 2 (3), the RAG-1 gene of ES cells was deleted, and a RAG-1 deletion allele was formed. Ear DNA is prepared from a pup mouse obtained by mating (RAG-1 ^+/- XVα14tg) F1 mice obtained by such a method, and RAG-1 ^{− / −} / Vα14tg mouse is selected by PCR method did.
(2) On the other hand, FIG. 3 shows a construct for producing a Vβ8.2 TCR gene transgenic mouse (Vβ8.2tg mouse). A cosmid library was prepared from BALB / c mice using the cosmid vector pTCF. A 35 kb long genomic DNA construct with the reconstructed Vβ8.2DβJβ2.4 gene was used as a construct to create Vβ8.2tg mice. The reconstructed Vβ8.2DβJβ and Cβ parts are shown enlarged in FIG. This Vβ8.2tg mouse (provided by Dr. Boehmer, Uematsu Y, Ryser S, Dembic Z, et al. Intransgenic mechanical the instrogen gen ss. Were mated with RAG-1 ^{− / −} mice, and RAG-1 ^{− / −} /Vβ8.2tg mice were prepared in the same manner as described above.
(3) The RAG-1 ^{− / −} / Vα14tg mouse obtained above and the RAG-1 ^{− / −} /Vβ8.2 tg mouse were mated, ear DNA was prepared for the obtained mouse, and RAG was prepared by PCR method. -1 gene deletion and coexpression of Vβ8.2tg / Vα14tg were confirmed, and RAG-1 ^{− / −} /Vβ8.2tg/Vα14tg mice were selected. The primers used for each PCR are shown below.

(C) Using the spleen mononuclear cells of the prepared RAG-1 ^{− / −} /Vβ8.2tg/Vα14tg mouse, compared with the previously reported NKT cells, the surface trait and functionally equivalent It was evaluated as follows.
Preparation of Spleen Cells Three types of C57BL / 6 mice, RAG-1 ^{− / −} /Vβ8.2 tg mice, RAG-1 ^{− / −} /Vβ8.2 tg ^/ Vα14 tg mice, all 6-8 weeks old female mice were used. After removing the spleen of each mouse, it was homogenized using a slide glass, and ACK lysis buffer (0.15M NH ₄ Cl, 1.0 mM KHCO ₃ , 0.1 mM Na ₂ EDTA, pH 7.2-7.4). Was used for hemolysis treatment to prepare spleen mononuclear cells. In the cell proliferation test and cytotoxicity test described below, spleen mononuclear cells were prepared to 2 × 10 ⁶ / ml with RPMI 1640 supplemented with 10% fetal calf serum (FCS) and used for the subsequent experiments.

FACS analysis Spleen mononuclear cells (5-10 × 10 ⁵ ) prepared from each mouse were incubated with 10 μg / ml anti-CD32 / 16 (2.4G2) antibody to inhibit Fc-mediated nonspecific antibody binding. Various cell staining was performed. For cell staining, three-color staining using the following monoclonal antibodies was used.
Cyclochrome avidin, Fluorescein isothiocyanate (FITC) binding anti-TCRβ (H57-597), FITC binding CD3-ε (145-2C11), FITC binding CD45RB (16A), FITC binding CD62L (MEL-14), FITC binding CD4 ( RM4-5), Phycoerythrin (PE) -conjugated anti-Vα14 (CMS-1), PE-conjugated CD8 (53-6.7), Vithion-conjugated anti-NK1.1 (PK136)
To prove the specificity of Vα14 staining, cells were pretreated with an excess amount of unlabeled anti-Vα14 and then reacted with PE-conjugated anti-Vα14 (Cold blocking). For FACS analysis, EPICS-XL (Coulter Electronics, Hialeah, FL) was used. The result is shown in FIG. All analysis diagrams were developed with CD3 / NK1.1 because the CD3 molecule forms a complex with the TCR and is an essential structure for T cell signaling, ie, function. This is because it can also prove sex T lymphocytes.
From the FACS analysis chart, it can be seen that Vα14NKT cells are present in CD3-positive NKT cells in C57BL / 6 mice (upper row in FIG. 4).
In RAG-1 ^{− / −} /Vβ8.2tg mice (middle panel in FIG. 4), CD3-positive cells did not appear. Since the CD3 negative NK1.1 positive cells of this mouse were TCRβ positive, it was found that they are not NK cells but progenitors of NKT cells.
In RAG-1 ^{− / −} /Vβ8.2tg/Vα14tg mice (lower part of FIG. 4), NK1.1-positive CD3-positive cells appeared, and the cells were Vα14-positive. That is, the only mature T cells that could be found in RAG-1 ^{− / −} /Vβ8.2tg/Vα14tg mice were Vα14 positive NKT cells. Moreover, since the CD3-negative NK1.1 positive cells of this mouse were also TCRβ positive, it was found that they are not NK cells but progenitor cells of NKT cells.
Therefore, on the surface trait, the RAG-1 ^{− / −} /Vβ8.2tg/Vα14tg mouse was considered to be the target NKT cell.
Next, whether or not Vα14-positive NKT cells of RAG-1 ^{− / −} /Vβ8.2tg/Vα14tg mice are functionally equivalent to conventional NKT cells was examined below.

Cell proliferation ability and IL-4, IFN-γ production performance RAG-1 ^{− / −} /Vβ8.2tg/Vα14tg Maturated lymphocytes, ie, CD3 positive cells are only Vα14 positive NKT cells as described above.
Therefore, it was examined whether the cells could actually proliferate by stimulation via CD3.
On the other hand, conventional NKT cells are typical IL-4 supplying cells and also produce IFN-γ. Therefore, it was examined whether IL-4 and IFN-γ can be produced under stimulation via CD3.
Anti-CD3ε antibody (145-2C11) was prepared in PBS to 100 μg / ml, 10 μg / ml, 1 μg / ml, and each anti-CD3ε antibody solution 30 μl 96-well round bottom cell culture plate (Nunc, Roskilde, Denmark) After incubation at 37 ° C. for 90 minutes, the plate was washed twice with cold PBS to prepare an anti-CD3ε antibody-immobilized plate. On this plate, 100 μl (2 × 10 ⁵ ) of spleen cell suspension of RAG-1 ^{− / −} /Vβ8.2 tg ^/ Vα14 tg mouse prepared to 2 × 10 ⁶ / ml was added to each well at 37 ° C., 5% CO ₂ . Cultured in the environment for 3 days. 100 μCi / well of ³ H-thymidine (Amersham) was added for 6 hours in the latter half of the culture, and the uptake of ³ H-thymidine was measured with a liquid scintillation counter to perform a cell proliferation test. The results are shown in FIG.
In the same system, culture supernatants were collected 7 hours, 24 hours, and 48 hours after the start of culture, and IL-4 and IFN-γ producing ability was examined. The results are shown in FIG.
As shown in the figure, in the spleen cells of RAG-1 ^{− / −} /Vβ8.2tg/Vα14tg mice, the cell proliferation response was clearly induced by stimulation with anti-CD3ε antibody, and significant IL-4 and IFN-γ Production was seen. That is, it was found that in RAG-1 ^{− / −} /Vβ8.2tg/Vα14tg mice, the CD3 complex expressed only in Vα14NKT cells functions as an effective signaling molecule.

Cytotoxicity test NKT cells have been reported to have a cytotoxic function by the perforin system or Fas-Fas ligand system. Cytotoxic activity against tumor cell lines was measured as follows using spleen cells of RAG-1 ^{− / −} /Vβ8.2tg/Vα14tg mice as effector cells.
150 mg / mouse of poly (I: C) (Pharmacia Biotech) was intraperitoneally administered to RAG-1 ^{− / −} /Vβ8.2tg/Vα14tg mice, and after 18 hours, the spleens of the mice were removed and homogenized. Hemolysis was performed using ACK lysis buffer (0.15M NH ₄ Cl, 1.0 mM KHCO ₃ , 0.1 mM Na ₂ EDTA, pH 7.2-7.4) to obtain effector cells. NK-sensitive YAC-1 cells were selected as target cells, labeled with 100 μCi of ⁵¹ C-chromium per 5 × 10 ⁶ cells at 37 ° C. for 1 hour, washed 3 times with RPMI 1640, and then the target cells. Used as. The reaction was performed in a 96-well round-bottom cell culture plate (Nunc, Roskilde, Denmark), and the ratio of the number of effector cells to the number of target cells (E / T ratio) for 1 × 10 ⁴ cells / well of YAC-1 cells. 51, 25, 25 RAG-1 ^{− / −} /Vβ8.2 tg ^/ Vα14 tg mouse spleen mononuclear cells were added, reacted at 37 ° C., 5% CO ₂ for 4 hours, and released ⁵¹ C-chromium Was measured with a γ counter. As a control experiment of poly (I: C), spleen mononuclear cells of mice in which 100 μl of PBS was intraperitoneally administered were used. The results are shown in FIG.
Spleen mononuclear cells of RAG-1 ^{− / −} /Vβ8.2tg/Vα14tg mice had NK activity from the naïve state. Furthermore, its NK activity was enhanced by administration of poly (I: C). Among the spleen cells of RAG-1 ^{− / −} /Vβ8.2tg/Vα14tg mice, cells considered to have NK activity are considered to be NK1.1 positive cells, ie, NKT cells only. Therefore, it was found that Vα14NKT cells of RAG-1 ^{− / −} /Vβ8.2tg/Vα14tg mice have cytotoxic activity in the same manner as conventional NKT cells.

を各１０μＭの濃度で、０．１μｇのＤＮＡを鋳型として、１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ９．０）、７５ｍＭ ＫＣｌ、１．５ｍＭ ＭｇＣｌ_２、２００μＭ ｄＮＴＰ Ｍｉｘ、５％ＤＭＳＯの条件で５Ｕ ＡｍｐｌｉＴａｑＤＮＡポリメラーゼ（Ｐｅｒｋｉｎ−Ｅｌｍｅｒ、Ｎｏｒｗａｌｋ、ＣＴ）を加え、総量５０μｌでＤＮＡ Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ（Ｍｏｄｅｌ ＰＪ２０００、Ｐｅｒｋｉｎ−Ｅｌｍｅｒ）を用いて、９４℃で５分間反応させたのち、９４℃１分、６２℃１分、７４℃２分のサイクルを３０回繰り返した。反応後のＰＣＲ産物は、１０μｌを０．８％アガロースゲル上で電気泳動（１００Ｖ、２５分間）を行い、１．８ｋｂのバンドを０．５μｇ／ｍｌのｅｔｈｉｄｉｕｍ ｂｒｏｍｉｄｅで染色して検出した（図９参照）。
ＤＮＡブロット法は、ＤＮＡ２０μｇを制限酵素ＨｉｎｄＩＩＩで１６時間反応させて切断したものを、０．８％アガロースゲル上で電気泳動（３０Ｖ、１６時間）を行い、ゲルからナイロン膜にＤＮＡ断片をうつしとり、放射線同位元素Ｐ^３２で標識したＤＮＡプローブ（プローブＡとプローブＢ（図８（２）、（３）に示したように、プローブＡはＳａｌ１／Ｓａｌ１断片、プローブＢはＳａｌ１／Ｃｌａ１断片である）と特異的に結合させ、バイオイメージアナライザー（ＢＡＳｔａｔｉｏｎ、富士フィルム株式会社、東京、日本）を用いることで、特定の長さのＤＮＡ断片（変異したＪα２８１遺伝子座は３．６ｋｂのバンドになる）として検出できる（図１０参照）。
上記で得られた５個のＥＳ細胞クローンを用いて、ＢＤＦ２マウスをホストにして凝集キメラ法により次のようにキメラマウスを作成した。１０−２０個のＥＳ細胞塊と透明帯を取り除いたＢＤＦ２マウス８細胞期胚をＭ１６培地（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）内で一晩培養してキメラ桑実胚へと分化させ、それを偽妊娠状態のＩＣＲマウス雌の子宮内へ戻すことで、９８種類のキメラマウスが得られ、そのうち２０種類が生殖細胞キメラであった。
キメラマウスとＣ５７ＢＬ／６マウスとの交配により対立遺伝子のうちの一方のＪα２８１遺伝子を欠いたマウス（ヘテロ接合体；＋／−と表示）を得て、これを交配することで両方のＪα２８１遺伝子を欠いたマウス（ホモ接合体；−／−と表示）を得た。
生まれたマウスの尾の一部よりＤＮＡを抽出し、ＰＣＲ法およびＤＮＡブロット法により遺伝子型を確認した。
ＰＣＲ法は、上記と同じ条件で、プライマー１とプライマー３で１．８ｋｂの変異したＪα２８１遺伝子座を検出し、プライマー１とプライマー２

で１．５ｋｂの野生型のＪα２８１遺伝子座を検出した（図９）。ＤＮＡブロット法も上記と同じ条件で、変異したＪα２８１遺伝子座は３．６ｋｂのバンド、野生型のＪα２８１遺伝子座は７．２ｋｂのバンドとして検出した（図１０）。 Example 2 Generation of NKT cell-deficient mice DNA clones containing the Jα281 gene fragment were screened using a genomic DNA library of 129SV mice. A vector for generating a gene-deficient mouse (hereinafter referred to as a targeting vector) is a 1.2 kb SalI / ClaI fragment present on the 5 ′ side of the Jα281 gene fragment in the short arm and a 12 kb located on the 3 ′ side of the Jα281 gene fragment in the long arm The exon portion of the Jα281 gene was replaced with a reverse PGK-neo-polyA cassette (provided by Dr. Shigemi Itohara, Kyoto University). It was created by deleting completely (Fig. 8 (1)). Note that other adjacent Jα gene fragments are not affected by this deletion. In order to reduce the insertion of the targeting vector into random genomic DNA, a DTA cassette (GIBCO BRL, Gaithersburg, MD) was introduced on the 5 ′ side of the short arm.
R1 ES cells (provided by Dr. A. Nagy, Mountsinai Hospital, FIG. 8 (2)) using the targeting vector electroporation method (550 μF, 270 mV, Gene Pulser II ^™ , Bio-Rad Laboratories, Hercules, Calif.) The gene was introduced.
R1 medium (Dallbeco MEM, 2 mM 1-glutamine, 50 μg / ml streptomycin, 50 μg / ml penicillin, 10 μM βME, 20000 U / ml LIF (ESGRO; GIBCO BRL), 0.1 mM non-essential amino acid with 200 μg / ml G418 (GIBCO BRL) And 20% fetal bovine serum (FCS)), and 45 G418-resistant ES cell colonies were isolated 8-10 days after the start of culture.
DNA was extracted from G418-resistant ES cells, and 5 clones of ES cells in which homologous recombination occurred at the Jα281 locus were selected by the following PCR method and DNA blot method.
The PCR method is shown in FIG.

5 U AmpliTaq DNA polymerase (Perkin) under the conditions of 10 mM Tris-HCl (pH 9.0), 75 mM KCl, 1.5 mM MgCl ₂ , 200 μM dNTP Mix, 5% DMSO using 0.1 μg of DNA as a template. Elmer, Norwalk, CT) was added and reacted at 94 ° C. for 5 minutes using DNA Thermal Cycler (Model PJ2000, Perkin-Elmer) in a total volume of 50 μl, then 94 ° C. for 1 minute, 62 ° C. for 1 minute, 74 ° C. The 2 minute cycle was repeated 30 times. After the reaction, 10 μl of the PCR product was electrophoresed on a 0.8% agarose gel (100 V, 25 minutes), and a 1.8 kb band was stained with 0.5 μg / ml of ethanol bromide and detected (FIG. 9).
In the DNA blotting method, 20 μg of DNA was digested with the restriction enzyme HindIII for 16 hours and cleaved, and electrophoresed on a 0.8% agarose gel (30 V, 16 hours) to transfer the DNA fragment from the gel to a nylon membrane. , radioisotope ^{P 32-labeled} DNA probe (probe a and probe B (FIG. 8 (2), as shown in (3), probe a Sal1 / Sal1 fragment, probe B is Sal1 / Cla1 fragment ), And using a bioimage analyzer (BAStation, Fuji Film Co., Ltd., Tokyo, Japan), a DNA fragment of a specific length (the mutated Jα281 locus becomes a 3.6 kb band) (See FIG. 10).
Using the 5 ES cell clones obtained above, a chimeric mouse was prepared as follows by the agglutination chimera method using BDF2 mouse as a host. BDF2 mouse 8-cell stage embryos from which 10-20 ES cell masses and zona pellucida have been removed are cultured overnight in M16 medium (Sigma Chemical Co., St. Louis, MO) to differentiate into chimeric morulas. By returning it to the uterus of female ICR mice in a pseudopregnant state, 98 types of chimeric mice were obtained, 20 of which were germ cell chimeras.
By crossing a chimeric mouse with a C57BL / 6 mouse, a mouse lacking one of the alleles Jα281 gene (heterozygote; indicated as +/−) was obtained and crossed to obtain both Jα281 genes. Mice lacking (homozygotes; labeled − / −) were obtained.
DNA was extracted from a part of the tail of the born mouse, and the genotype was confirmed by PCR and DNA blotting.
The PCR method detects a 1.8 kb mutated Jα281 locus with primer 1 and primer 3 under the same conditions as above, and primer 1 and primer 2

A 1.5 kb wild-type Jα281 locus was detected (FIG. 9). Under the same conditions as described above, the DNA blot method also detected a mutated Jα281 locus as a 3.6 kb band and a wild type Jα281 locus as a 7.2 kb band (FIG. 10).

About the created Jα281 gene-deficient mouse (− / −), the surface traits and functions were compared with the littermate control mouse (+ / +) using a mononuclear cell suspension as follows.
FACS analysis A mononuclear cell suspension was prepared from the thymus, spleen, bone marrow and liver of Jα281 gene-deficient mice (− / −) and littermate control mice (+ / +) as follows. The thymus and spleen were shredded with scissors in a petri dish containing a small amount of medium (PBS containing 10% FCS) and then crushed between glass slides.
For the bone marrow, the femur and tibia were removed from the hind limb of the mouse, the upper end and the lower end were cut off, the medium was placed in a syringe with a 23G needle, and the bone marrow was pushed out by inserting a needle into one stump in the petri dish.
The liver is shredded in a petri dish containing 15 ml of PBS, added with 400 U / ml collagenase, reacted at 37 ° C. for 20 minutes, then crushed on a # 200 mesh stainless steel mesh to give a pellet of 50 U / The suspension was well suspended in 15 ml of 33% Percoll (Pharmacia, Uppsala, Sweden) plus ml heparin + PBS, and then centrifuged at 2000 rpm for 10 minutes. In addition, the mixed red blood cells were removed with ACK lysis buffer (0.15M NH ₄ Cl, 1 mM KHCO ₃ , 0.1 mM Na ₂ EDTA, pH 7.3).
After binding 1-2 × 10 ⁶ cells with a monoclonal antibody against FcγII / III receptor (2.4G2; PharMingen, San Diego, Calif.) To block nonspecific antibody binding, fluorescent dye Fluorescein isothiocyanate (FITC) labeling Anti-T cell receptor β chain antibody (H57-597; PharMingen) and Phycoerythrin (PE) labeled anti-NK1.1 antibody (PK136; PharMingen) were double-stained with EPICS-XL (Coulter, Hialeah, FL) Analysis was performed. The result is shown in FIG.
In mice lacking the Jα281 gene, NK cells and T cells were observed in both thymus and peripheral tissues (spleen, bone marrow and liver), but only NKT cells were defective.

As mentioned in IL-4 production example 1, NKT cells have been reported to be a major production cells of IL-4 in vivo. Therefore, the IL-4 production ability of the NKT cell-deficient mice prepared above was examined. Anti-CD3ε antibody (2C11: 1.5 μg) was intravenously injected into mice, and after 90 minutes, the spleen was removed and RNA was extracted, and the expression of IL-4 at the mRNA level was examined by RNAase protection method and RT-PCR method It was. Furthermore, the spleen cells were cultured for 48 hours on the immobilized anti-CD3 antibody, and IL-4 in the supernatant was measured by ELISA.
As a result, NKT cell-deficient mice did not produce IL-4.

Schematic diagram of Vα14TCR construct: pHβAPr-Tsα2-neo. The restriction enzymes involved in the restriction site indicated by each symbol are as follows. B: BglII, E: EcoRI, S: SalI, H: HindIII, P: PvuI. The figure which shows the method for destroying RAG-1 gene. (1) represents a knockout construct. (2) represents an ES cell genome containing the RAG-1 gene. (3) represents the RAG-1 deletion allele. X between (1) and (2) indicates the position where homologous recombination occurs. Construct for making Vβ8.2tg mice. FACS analysis diagram of splenic mononuclear cells of each mouse. Upper row: C57BL / 6 mice, middle row: RAG-1 ^{− / −} /Vβ8.2 tg mice, lower row: RAG-1 ^{− / −} /Vβ8.2 tg ^/ Vα14 tg mice. The graph which shows the cell growth ability of a RAG-1 ^-/-/ V (beta) 8.2tg ^/ V (alpha) 14tg mouse | mouth. The graph which shows the IL-4 and IFN-gamma production ability of a RAG-1 < ^-/-/ V (beta) 8.2tg ^/ V (alpha) 14tg mouse | mouth. The graph which shows the cytotoxic activity of RAG-1 ^-/-/ V (beta) 8.2tg ^/ V (alpha) 14tg mouse | mouth. DNA construct for homologous recombination for disruption of Jα281 gene, genome map schematic diagram. (1) represents a DNA construct restriction enzyme map for homologous recombination. (2) shows a genome map and a restriction enzyme map on the side subjected to gene disruption including the Jα281 gene. (3) represents the genome map and restriction enzyme map of the Jα281 gene deletion allele. The restriction enzymes involved in the restriction site indicated by each symbol are as follows. C: ClaI, H: HindIII, S: SalI, N: NotI, X: XholI. The photograph of the gel which electrophoresed the DNA obtained by PCR method. Photo of gel showing electrophoresis by DNA blotting. FACS analysis diagram of mononuclear cells of thymus, spleen, bone marrow and liver of Jα281 gene-deficient mice (− / −).

Claims (4)

  Mice deficient in NKT cells.   The mouse according to claim 1, wherein the NKT cell defect is caused by a uniform Vα14Jα281α chain defect.   The mouse according to claim 2, wherein the NKT cell defect is caused by a defect in the Jα281 gene.   The mouse according to claim 3, wherein a neomycin resistance gene is inserted into an exon of the Jα281 gene.

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