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

ES 和 EG 细胞向种系诱导分化的研究。

• 批准号: 11460132
• 负责人: TACHI Chikashi
• 金额: $ 9.41万
• 依托单位: AZABU UNIVERSITY
• 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 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11460132/
• 关键词: ES cells; PGC; Germ line cells; SCF; Feeder cells; 4C9; 2C9; Goats; EG細胞; 始原生殖細胞; 4C9抗体; 2C9抗体; 卵黄嚢細胞; ヤギ; ブタ

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

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 ...更多信息 （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

项目成果

Inuzuka, H., Yamanouchi, K., Tachi, C., Tojo, H.: "Expression of milk protein gene is induced directly by exogenous STATS without prolactin-mediated signal transduction in transgenic mice."Mol Repro Dev. 54. 121-125 (1999)

Inuzuka, H.、Yamanouchi, K.、Tachi, C.、Tojo, H.：“在转基因小鼠中，乳蛋白基因的表达是由外源 STATS 直接诱导的，无需催乳素介导的信号转导。”Mol Repro Dev.

Tada, T., Obata, Y., Tada, M., Goto, Y., Nakatsuji, N., Tan, S.-S., Kono, T.and Takagi, N.: "Imprint switching for non-random X-chromosome inactivation during mouse oocyte growih."Development. 127. 3101-3105 (2000)

Tada, T.、Obata, Y.、Tada, M.、Goto, Y.、Nakatsuji, N.、Tan, S.-S.、Kono, T. 和 Takagi, N.：“非随机的印记切换

Tachi, C.: "Analysis of gene functions. In : "Analysis of Genome and Strategies for Breeding in Farm Animals", ed. by Committee for Symposium on Genetics and Animal Breeding"Association for Animal Biotechnology, Tokyo. 109-115 (2000)

Tachi, C.：“基因功能分析。见：“农场动物基因组分析和育种策略”，由东京动物生物技术协会遗传学和动物育种研讨会委员会编辑。

B.B.Seo.C.H.Kim,K.Yamanouchi,M.Takahshi,T.Sawasaki C.Tachi,H.Tojo: "Co-injection of restriction enzyme with foreign DNA into the pronucleus for elevating production efficiencies of transgemic animals."Anim.Reprod.Sci.. 63. 113-122 (2000)

B.B.Seo.C.H.Kim,K.Yamanouchi,M.Takahshi,T.Sawasaki C.Tachi,H.Tojo：“将限制性酶与外源 DNA 共同注射到原核中，以提高转基因动物的生产效率。”Anim.Reprod

K.Miyoshi,N.Kashiwazaki,E.Sato: "Development of porcine nuclear transfer embryos reconstituted with blastocysts-derived cells and enucleated oocytes."Asian-Aus. J.Anim.Sci.. Supplement13. 201 (2000)

K.Miyoshi、N.Kashiwazaki、E.Sato：“用囊胚衍生细胞和去核卵母细胞重建猪核移植胚胎的开发。”亚洲-澳大利亚。