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

利用分子遗传学生产猪体细胞克隆动物的研究

基本信息

批准号：
11556051
负责人：
NAITO Kunihiko
金额：
$ 7.42万
依托单位：
THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11556051/
关键词：
porcine oocytes somatic cell clone enucleation nuclear transfer MPF MRPK protein synthesis Rb

项目摘要

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

本研究的最初目的是生产猪体细胞克隆。然而，在2000年，猪体细胞克隆已在其他实验室生产。因此，本文对克隆动物的材料卵母细胞和早期胚胎的生理机制和发育调控因素进行了分析，以期提高克隆动物生产的效率。(1)首先，我研究了通过去除纺锤体和交换体细胞核的方法去除成熟促进因子（MPF）和MAP激酶（MAPK）的可能性。我能够证明MPF的去除非常小，可能对胚胎发育没有影响，但是大约20%的MAPK被这个过程去除，可能会影响胚胎发育。(2)其次，研究了这些因子在重组卵母细胞中是否定位于被替换的体细胞染色体上。我发现MPF和MAPK定位于 ...更多信息艾德只在微管蛋白定位的纺锤体上表达，而在微管蛋白非定位的纺锤体上不表达。此外，由于微管蛋白定位纺锤体的出现频率与原核的形成频率一致，因此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)接下来，我分析了在减数分裂恢复前后发生的蛋白质合成模式交换的调节因素。本研究以MPF和MAPK为调控因子，将MPF和MAPK的mRNA或反义RNA注入卵母细胞，人为改变其活性。结果表明，MPF调控蛋白质合成模式的转换，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，该细胞周期没有间隙期，仅快速重复S期和M期。本研究以S期抑制性调节因子Rb蛋白的缺失为因素，从mRNA和蛋白水平检测了Rb蛋白在早期胚胎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缺失的，并通过将Rb表达载体注射到胚胎中抑制发育证实了Rb的参与，这些结果不仅是哺乳动物而且是所有物种的首次报道，为提高克隆动物生产效率提供了基础数据。少