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

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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.