Jakobovits A, Moore A L, Green L L, Vergara G J, Maynard-Currie C E, Austin H A, Klapholz S
Cell Genesys Inc., Foster City, California 94404.
Nature. 1993 Mar 18;362(6417):255-8. doi: 10.1038/362255a0.
Introduction of DNA fragments, hundreds of kilobases in size, into mouse embryonic stem (ES) cells would greatly advance the ability to manipulate the mouse genome. Mice generated from such modified cells would permit investigation of the function and expression of very large or crudely mapped genes. Large DNA molecules cloned into yeast artificial chromosomes (YACs) are stable and genetically manipulable within yeast, suggesting yeast-cell fusion as an ideal method for transferring large DNA segments into mammalian cells. Introduction of YACs into different cell types by this technique has been reported; however, the incorporation of yeast DNA along with the YAC has raised doubts as to whether ES cells, modified in this way, would be able to recolonize the mouse germ line. Here we provide, to our knowledge, the first demonstration of germ-line transmission and expression of a large human DNA fragment, introduced into ES cells by fusion with yeast spheroplasts. Proper development was not impaired by the cointegration of a large portion of the yeast genome with the YAC.
将数百千碱基大小的DNA片段导入小鼠胚胎干细胞，将极大地提升操控小鼠基因组的能力。由这种经过修饰的细胞培育出的小鼠，将有助于研究非常大的基因或定位粗略的基因的功能及表达情况。克隆到酵母人工染色体（YAC）中的大型DNA分子在酵母细胞内是稳定的且可进行基因操作，这表明酵母细胞融合是将大型DNA片段导入哺乳动物细胞的理想方法。已有报道通过该技术将YAC导入不同细胞类型；然而，酵母DNA与YAC一同整合，引发了人们对于以这种方式修饰的胚胎干细胞能否重新定殖于小鼠生殖系的质疑。在此，据我们所知，我们首次证明了通过与酵母原生质球融合导入胚胎干细胞的大型人类DNA片段能够进行种系传递并表达。酵母基因组的很大一部分与YAC共整合并未损害小鼠的正常发育。
