Pachnis V, Pevny L, Rothstein R, Costantini F
Department of Genetics and Development, Columbia University, New York, NY 10032.
Proc Natl Acad Sci U S A. 1990 Jul;87(13):5109-13. doi: 10.1073/pnas.87.13.5109.
To test the feasibility of transferring yeast artificial chromosomes (YACs) into mammalian cells, we modified a YAC that carries approximately 450 kilobases (kb) of human DNA, by inserting a neomycin-resistance gene. Saccharomyces cerevisiae cells carrying this YAC were fused by polyethylene glycol to mouse L cells and G418-resistant colonies were obtained. A high percentage of these clones contained virtually intact YAC sequences as revealed by "Alu fingerprint" analysis and restriction enzyme analysis using pulsed-field gel electrophoresis. Furthermore, the YAC sequences were stably integrated into the mouse chromosomes, as shown by in situ hybridization and by the stability of the G418 resistance. These results establish that large segments of the mammalian genome, cloned in yeast, can be efficiently transferred into cultured mammalian cells.
为了测试将酵母人工染色体（YACs）转入哺乳动物细胞的可行性，我们通过插入一个新霉素抗性基因，对携带约450千碱基（kb）人类DNA的YAC进行了改造。携带这种YAC的酿酒酵母细胞通过聚乙二醇与小鼠L细胞融合，并获得了对G418抗性的菌落。如通过“Alu指纹”分析和使用脉冲场凝胶电泳的限制性酶切分析所揭示的，这些克隆中有很大比例实际上含有完整的YAC序列。此外，原位杂交和G418抗性的稳定性表明，YAC序列已稳定整合到小鼠染色体中。这些结果证明，克隆于酵母中的大片段哺乳动物基因组能够有效地转入培养的哺乳动物细胞。
