RAT RESOURCE AND RESEARCH CENTER: CLONING TECHNOLOGY

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。利用胚胎干细胞进入实验动物的生殖系对我们更好地理解基因功能非常重要。尽管这种方法很有价值，但它对老鼠以外的物种无效。虽然胚胎干细胞已经从包括大鼠在内的多种物种中分离出来，但迄今为止，这些细胞还没有被证明具有足够的多能性，无法产生种系嵌合体。另一种生殖细胞的方法是核移植。利用培养细胞作为遗传物质来源指导兔、牛、羊去核卵母细胞的胚胎发育的最新进展，为在大鼠体内进行同源重组提供了途径。我们已经建立了大鼠卵母细胞去核、注射卵丘细胞和遗传标记的大鼠胚胎成纤维细胞的程序，从而导致植入前的高级发育。如果证明通过这种方法恢复活产是可行的，那么敲除成纤维细胞中的基因就可以用来获得突变的大鼠。核移植产生的大鼠本质上是培养细胞的来源菌株，无需后续繁殖，因此原则上允许使用特定菌株的大鼠，对给定的实验最有意义。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The use of embryonic stem cells to gain access to the germline of experimental animals has been important to our greater understanding of gene function. As valuable as this approach is, it has not worked for species other than mouse. While ES cells have been isolated from a variety of species including the rat, the cells have not so far proven to be sufficiently pluripotent to allow the creation of germline chimeras from them. Another approach to the germline, though, is nuclear transfer. Recent advances in the use of cultured cells as a source of genetic material to direct embryonic development of enucleated oocytes in rabbit, cow, and sheep offer an approach to homologous recombination in the rat. We have established procedures for the enucleation of rat oocytes, the injection of cumulus cells and of genetically marked embryonic fibroblasts from the rat resulting in advanced preimplantation development. If it proves feasible to recover live births from this approach, then knocking-out genes in the fibroblasts can be used to obtain mutant rats. Nuclear transfer results in a rat which is essentially the strain of origin of the cultured cells without subsequent breeding and therefore allows, in principle, the use of particular strains of rat for which given experiments make the most sense.