Basic study for the production of nock-out pig by nucleiar transfer using recombinant somatic cell

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Inositol 1,4,5-trisphosphate (IP3) is considered to be important for activation of mammalian oocytes at the time of fertilization, and that the activation induces a rise in intracellular Ca^<2+> concentration. Therefore, IP3 can be useful as an artificial activator of oocytes. In this study, we injected IP3 into porcine matured oocytes and demonstrated that injected IP3 promotes the development of the matured oocytes and that their developmental ability is positively correlated with the rise in Ca^<2+> concentration induced by IP3. Naturally, mammalian oocytes are activated with sperm penetration, and it is suggested that some unknown sperm factor must activate the oocytes. For several years, it has been argued what is a real sperm factor to activate oocytes. Recently, phospholipaseC(PLC)ζ cDNA is cloned and is expected to be the most useful candidate to activate the oocytes. We originally succeed to clone the porcine PLCζcDNA. In addition to these activation works, and finally in vitro developmental study of porcine nuclear transfer embryos from foreign gene-transfected somatic cells was investigated. We used enhanced green fluorescent protein(EGFP) gene as a foreign gene maker. The result indicated that EGFP gene transfected into oocytes has no deleterious effect in the embryonic development to blastocyst stage. However, the clear transfer fetus which is expressing EGFP gene could not be obtained so far.