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

我们以前曾报道的雌激素依赖性输卵管特异性糖蛋白（OGP）基因在小鼠中的分离和部分特征。虽然已经表明，OGP，发现在输卵管液从各种哺乳动物物种，可能参与受精过程，包括精子功能和配子相互作用，其在体内的生理意义仍然存在争议。本研究的目的是建立胚胎干（ES）细胞系，其缺乏大部分的小鼠OGP编码区，用于生产OGP敲除小鼠，以阐明该分子的生理功能。从TT 2文库中分离的基因组OGP DNA用于构建靶向载体。其中，编码OGP的N-末端部分的外显子I-VI被新霉素抗性基因取代，并且单纯疱疹胸苷激酶基因在3 '末端融合。通过电穿孔将靶向载体插入TT 2细胞中，然后通过G418和更昔洛韦的选择富集同源重组事件。通过突变特异性PCR和Southern杂交鉴定突变克隆。将三个携带OGP突变的独立突变的ES克隆用于通过8细胞注射产生突变小鼠。利用胚胎DNA的基因组特征显示，已经建立了起源于这些突变ES细胞的杂合突变小鼠。这些小鼠将是阐明OGP在体内受精过程中的意义的一个非常强大的工具。

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.