Studies on the Induction of Differentiation in ES and EG cells into the Germ Line.

利用体细胞核移植技术生产转基因动物或克隆动物，已成为畜牧业中的一种实用手段，但仍存在许多问题。其中一个问题涉及未受精卵的可用性，这些卵子通常难以获得足够的数量。如果能在体外诱导XX核型的ES/EG细胞进入生殖系，并最终分化为卵子，将在动物生物技术，特别是大型哺乳动物生物技术的各个领域取得重大进展。本研究的目的是进行基础研究，探索开发使ES/EG细胞转化为生殖系的技术的可能性。在下文中，总结了实验结果。1)鼠ES细胞系的免疫细胞化学研究揭示了与4C 9（抗PGC标记蛋白的单克隆抗体）和2C 9反应的细胞的存在 关于我们 （抗鸡PGC的单克隆抗体）阳性，可能代表群体的生殖系细胞。在刺激PGC增殖的条件下培养4C 9/2C 9阳性ES细胞对这些细胞没有显著刺激。对已知在PGC中表达的vasa基因的结构和功能进行了分析。2)为了建立山羊PGC体外培养体系，作为牛科动物的模型，克隆了山羊SCF（别名：KL和MGF）cDNA，并用于建立表达膜结合型和可溶型cSCF的饲养细胞系。3)分析了在XX染色体核型的ES细胞中对X染色体Lyonization起关键作用的Xist及其反义基因Tsix的表达。证实了2个基因的表达。4)对猪卵母细胞体外成熟和体外受精的方法进行了研究。为核酶在家畜遗传操作中的应用做了基础研究。5)摘要近年来，ES/EG细胞向生殖系转化的研究进展迅速。目前的研究项目为解决这个问题提供了有价值的线索。少

In artyodactyls, the production of transgenic animals or clonal animals by means of nuclear transfer from somatic cells, is rapidly becoming practical means in animal industries despite many unsolved problems. One of such problems concerns with the availability of unfertilized ova which are often difficult to obtain in sufficient quantity. If ES/EG cells with the XX karyotype could be induced to enter into the germ line in vitro, and ultimately to differentiate into ova, an important progress will be made in various fields of animal biotechnology, especially those dealing with large mammals. The purpose of present studies is to carry out basic research exploring the possibility of developing techniques which enable the convrsion of ES/EG cells to the germ line. In the following, the results of experiments are summarized. 1) Immunocytochemical studies of a murine ES cell line revealed the presence of cells which reacted with 4C9 (monoclonal antibody against a PGC marker protein) and 2C9 … More (monoclonal antibody raised against chicken PGC) positively, probably respresenting germ line cells of the population. Culture of 4C9/2C9 positive ES cells under the conditions which stimulated PGC proliferation did not cause significant stimulation of these cell. Analysis of gene structure and functions of vasa which is knoown to be expressed in PGC was made. 2) In order to establslish an in vitro culture system for caprine PGC, as a model for bovidae animals in general, caprine SCF (synonyms : KL and MGF) cDNA was cloned, and usd for the establishement of the feeder cell lines where the membrane-bound as well as soluble form of cSCF are expressed. 3) ES cells with XX karyotype were analyzed for the expression of Xist and its antisense gene Tsix both of which play crucail roles in Lyonization of X chromosomes. The expression of the 2 genes was confirmed. 4) The IVM and IVF procedures for porcine oocytes were investigated. Basic studies were done for the application of ribozymes in the genetic manipulation of farm animals. 5) Summary. In recent years, rapid progress has been made in the studies on conversion of ES/EG cells to the germ line. The present research project yielded valuable clues to approach the problem. Less