RAT RESOURCE AND RESEARCH CENTER: CLONING TECHNOLOGY

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。利用胚胎干细胞获得实验动物的生殖系对于我们更好地了解基因功能非常重要。尽管这种方法很有价值，但除了老鼠之外，它对其他物种都不起作用。虽然ES细胞已经从包括大鼠在内的各种物种中分离出来，但到目前为止，这些细胞还没有被证明具有足够的多能性，可以从它们创建生殖系嵌合体。然而，另一种获得生殖系的方法是核移植。在利用培养细胞作为遗传物质来源指导兔、牛和羊去核卵母细胞胚胎发育方面的最新进展，为在大鼠中进行同源重组提供了一条途径。我们已经建立了去核大鼠卵母细胞、注射卵丘细胞和大鼠基因标记的胚胎成纤维细胞的程序，从而促进了植入前的发育。如果证明通过这种方法恢复活产是可行的，那么成纤维细胞中敲除的基因就可以用来获得突变大鼠。核移植产生的大鼠基本上是培养细胞的原始品系，没有随后的繁殖，因此，原则上允许使用特定品系的大鼠，对特定品系的实验是最有意义的。

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.