Studies for Production of Porcine Somatic Cell Clone Animals using Molecular Genetics

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The initial purpose of the present study is the production of porcine somatic cell clones. In 2000, however, the porcine somatic cell clones have been produced in other laboratories. Therefore I analyzed thereafter the physiological mechanisms and developmental regulation factors in oocytes and early embryos, which are the materials for the clone animal, in order to improve the efficiency of the clone animal production.(1) At First, I examined the possibility of the maturation promoting factor (MPF) and MAP kinase (MAPK) removal by the procedure for clone production, those are the removal of the spindle and the exchange of somatic nucleus. I was able to show that the MPF removal was very small and might have no effect on the embryo development, but that about 20 % of MAPK was removed by the process and might affect the embryo development.(2) Next, I studied whether these factors localized on the replaced somatic chromosomes in the reconstitute oocytes. I found that MPF and MAPK localiz … More ed only on the tubulin-localized-spindles but not on the tubulin-non localized-spindle. In addition, as the frequency of the tubulin-localized-spindle was well agreed with the frequency of pronucleus formation, I suggested that the oocytes having a tubulin-localized-spindle could form a pronucleus.(3) I analyzed next the factors regulating the protein-synthesis-pattern exchange, which occurred around meiotic resumption. I postulated MPF and MAPK as the factor and their mRNAs or antisense RNAs were injected into the oocytes in order to change their activity artificially. The results showed that the protein-synthesis-pattern exchange was regulated by MPF but not affected by MAPK acitvity.(4) Lastly, I investigated the regulator of the early embryo specific cell cycle, which has no gap phases and repeats only S phase and M phase rapidly. I postulated the absence ofRb protein, inhibitory regulator of S phase, as the factor and examined its expression levels during early embryo development at mRNA and protein levels. 1 found that Rb was actually absent between 4-cell stage and blastocyst stage, and its involvement was confirmed by the inhibition of development by the injection of Rb expression vector into embryos.These results are the first reports not only mammals but also all species, and might be useful to improve the efficiency of the clone animal production as basic data. Less