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

本研究的最初目的是生产猪体细胞克隆。然而，在2000年，猪体细胞克隆已经在其他实验室生产出来。因此，我随后分析了作为克隆动物材料的卵母细胞和早期胚胎的生理机制和发育调节因子，以提高克隆动物的生产效率。(1)首先考察了在克隆生产过程中去除成熟促进因子（MPF）和MAP激酶（MAPK）的可能性，即去除纺锤体和交换体细胞核。我能够证明MPF去除非常小，可能对胚胎发育没有影响，但是在这个过程中大约有20%的MAPK被去除，可能会影响胚胎发育。(2)接下来，我研究了这些因子是否定位于重建卵母细胞中被替换的体细胞染色体上。我发现MPF和MAPK只定位在微管蛋白定位纺锤体上，而不是在微管蛋白非定位纺锤体上。此外，由于微管蛋白定位纺锤体的频率与原核形成的频率非常一致，我认为具有微管蛋白定位纺锤体的卵母细胞可以形成原核。(3)分析了减数分裂恢复前后蛋白质合成模式交换的调节因素。我假设MPF和MAPK是因子，将它们的mrna或反义rna注射到卵母细胞中，人为地改变它们的活性。结果表明，蛋白合成模式交换受MPF调控，而不受MAPK活性的影响。(4)最后，研究了早期胚胎特异性细胞周期的调控因子，该周期没有间隙期，只快速重复S期和M期。假设S期抑制调节因子rb蛋白缺失为影响因素，并在mRNA和蛋白水平上检测其在胚胎早期发育过程中的表达水平。1发现Rb在4细胞期和囊胚期之间实际上是不存在的，通过将Rb表达载体注入胚胎抑制发育证实了Rb的参与。这些结果是首次在哺乳动物和所有物种中报道，并可能为提高克隆动物生产效率提供基础数据。少

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