ESTABLISHMENT OF GERM-LINE COMPETENT EMBRYONIC STEM CELLS LACKING MOUSE OVIDUCT-SPECIFIC GLYCOPROTEIN GENE

缺乏小鼠输卵管特异性糖蛋白基因的种系感受态胚胎干细胞的建立

• 批准号: 11671593
• 负责人: SHINKAI Yoichi
• 金额: $ 2.62万
• 依托单位: KYOTO UNIVERSIY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671593/
• 关键词: reproductive biology; OGP; knockout mouse; transgenic mouse; fertilization; mammal

We have previously reported the isolation and partial characterization of an estrogen-dependent oviduct-specific glycoprotein (OGP) gene in the mouse. Although it has been suggested that the OGP, found in the oviductal fluid from various mammalian species, may be involved in the fertilization process including sperm functions and gamete interaction, its physiological significance in vivo remains controversial. The objective of the present study was to establish embryonic stem (ES) cell lines which lack a large portion of the mouse OGP coding region for the production of an OGP knock out mouse to clarify the physiological function(s) of the molecule. Genomic OGP DNA, isolated from a library ofTT2 was used to construct a targeting vector. In this, exon I-VI encoding the N-terminal portion of OGP was replaced by the neomycin resistant gene, and the herpes simplex thymidine kinase gene was fused at the 3'-end. Targeting vector was inserted into TT2 cells by electroporation, then homologous recombination events were enriched by selection with G418 and gancyclovir. Mutant clones were identified by mutation specific PCR and Southern hybridization. Three independently mutated ES clones carrying OGP mutation were used to produce mutant mice by 8-cell injection. Genomic characterization using the embryonal DNA revealed that the heterozygous mutant mice originated from these mutant ES cells have been established. These mice will be a quite powerful tool to clarify the significance of OGP in the fertilization process in vivo.

我们之前报道了小鼠中雌激素依赖性输卵管特异性糖蛋白（OGP）基因的分离和部分表征。OGP存在于多种哺乳动物的输卵管液中，可能参与了包括精子功能和配子相互作用在内的受精过程，但其在体内的生理意义仍存在争议。本研究的目的是建立缺乏大部分小鼠OGP编码区的胚胎干细胞（ES）细胞系，以产生OGP敲除小鼠，以阐明该分子的生理功能。从tt2文库中分离的基因组OGP DNA用于构建靶向载体。其中编码OGP n端部分的I-VI外显子被新霉素耐药基因取代，单纯疱疹胸苷激酶基因在3'端融合。通过电穿孔将靶向载体插入TT2细胞中，用G418和更昔洛韦富集同源重组事件。通过突变特异性PCR和Southern杂交鉴定了突变克隆。3个携带OGP突变的独立突变ES克隆通过8细胞注射产生突变小鼠。利用胚胎DNA进行基因组鉴定表明，源自这些突变胚胎干细胞的杂合突变小鼠已经建立。这些小鼠将成为阐明OGP在体内受精过程中的重要意义的有力工具。

项目成果

Kurita, A., Takizawa, T., Takayama, T., Totsukawa, K., Matsubara, S., Shibahara, H., Orgebin-Crist, M.-C., Sendo, F., Shinkai.Y., Araki.Y.: "Identification, cloning and initial characterization of a novel mouse testicular germ cell-specific antigen"Biol.

栗田，A.，泷泽，T.，高山，T.，十津川，K.，松原，S.，芝原，H.，Orgebin-Crist，M.-C.，Sendo，F.，Shinkai.Y.，

Shinkai, Y. Satoh, H. Takeda, N. Fukuda, M. Chiba, E. Kato, T. Kuramochi, T. and Araki, Y.: "A testicular germ cell-associated serin-threonine kinase, MAK is despensable for sperm formation"Mol. Cell. Biol.. (in press). (2002)

Shinkai, Y. Satoh, H. Takeda, N. Fukuda, M. Chiba, E. Kato, T. Kuramochi, T. 和 Araki, Y.：“睾丸生殖细胞相关的丝氨酸-苏氨酸激酶，MAK 对于

Shinkai, Y., Satoh, H., Takeda, N., Fukuda, M., Chiba, E., Kato, T., Kuramochi, T., Araki, Y.: "A testicular germ cell-associated serine-threonine kinase, MAK is dispensable for sperm formation"Mol. Cell. Biol.. (2002)

Shinkai，Y.，Satoh，H.，Takeda，N.，Fukuda，M.，Chiba，E.，Kato，T.，Kuramochi，T.，Araki，Y.：“睾丸生殖细胞相关的丝氨酸-苏氨酸

Kurita A. Takizawa T. Takayama T, Totsukawa K. Matsubara S. Shibahara, H. Orgebin-Crist, M.-C. Sendo, F. Shinkai, Y. and Araki Y.: "Identification, cloning and initial characterization of a novel mouse testicular germ cell-specific antigen"Biol. Reprod..

栗田 A. 泷泽 T. 高山 T、十津川 K. 松原 S. 芝原、H. Orgebin-Crist、M.-C。