Transfer of a yeast artificial chromosome carrying human DNA from Saccharomyces cerevisiae into mammalian cells.
Transfer of a yeast artificial chromosome carrying human DNA from Saccharomyces cerevisiae into mammalian cells.
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DOI：
10.1073/pnas.87.13.5109
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发表时间：
1990-07
影响因子：
11.1
通讯作者：
V. Pachnis;L. Pevny;Rodney Rothstein;Frank Costantini
中科院分区：
文献类型：
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作者：
V. Pachnis;L. Pevny;Rodney Rothstein;Frank Costantini
为了测试将酵母人工染色体（YAC）转移到哺乳动物细胞中的可行性，我们通过插入新霉素抗性基因来修饰携带约450个内切酶（kb）的人类DNA的YAC。将携带该YAC的酿酒酵母细胞通过聚乙二醇与小鼠L细胞融合，获得了G418抗性克隆。这些克隆的高百分比包含几乎完整的YAC序列所揭示的“铝指纹”分析和限制性内切酶分析，使用脉冲场凝胶电泳。此外，YAC序列稳定整合到小鼠染色体中，如原位杂交和G418抗性的稳定性所示。这些结果表明，在酵母中克隆的哺乳动物基因组的大片段可以有效地转移到培养的哺乳动物细胞中。
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.