Reid L H, Shesely E G, Kim H S, Smithies O
Department of Pathology, University of North Carolina, Chapel Hill 27599.
Mol Cell Biol. 1991 May;11(5):2769-77. doi: 10.1128/mcb.11.5.2769-2777.1991.
We have investigated cotransformation in mammalian cells and its potential for identifying cells that have been modified by gene targeting. Selectable genes on separate DNA fragments were simultaneously introduced into cells by coelectroporation. When the introduced fragments were scored for random integration, 75% of the transformed cells integrated both fragments within the genome of the same cell. When one of the cointroduced fragments was scored for integration at a specific locus by gene targeting, only 4% of the targeted cells cointegrated the second fragment. Apparently, cells that have been modified by gene targeting with one DNA fragment rarely incorporate a second DNA fragment. Despite this limitation, we were able to use the cotransformation protocol to identify targeted cells by screening populations of colonies that had been transformed with a cointroduced selectable gene. When hypoxanthine phosphoribosyltransferase (hprt) targeting DNA was coelectroporated with a selectable neomycin phosphotransferase (neo) gene into embryonic stem (ES) cells, hprt-targeted colonies were isolated from the population of neo transformants at a frequency of 1 per 70 G418-resistant colonies. In parallel experiments with the same targeting construct, hprt-targeted cells were found at a frequency of 1 per 5,500 nonselected colonies. Thus, an 80-fold enrichment for targeted cells was observed within the population of colonies transformed with the cointroduced DNA compared with the population of nonselected colonies. This enrichment for targeted cells after cotransformation should be useful in the isolation of colonies that contain targeted but nonselectable gene alterations.
我们研究了哺乳动物细胞中的共转化及其在鉴定经基因打靶修饰的细胞方面的潜力。通过共电穿孔将位于不同DNA片段上的可选择基因同时导入细胞。当对导入的片段进行随机整合分析时，75%的转化细胞在同一细胞的基因组内整合了两个片段。当通过基因打靶对共导入片段之一在特定位点的整合进行分析时，只有4%的靶向细胞共整合了第二个片段。显然，经一个DNA片段基因打靶修饰的细胞很少整合第二个DNA片段。尽管有这个局限性，我们仍能够利用共转化方案，通过筛选用共导入的可选择基因转化的菌落群体来鉴定靶向细胞。当将次黄嘌呤磷酸核糖转移酶（hprt）靶向DNA与可选择的新霉素磷酸转移酶（neo）基因共电穿孔导入胚胎干细胞（ES细胞）时，从neo转化体群体中分离出hprt靶向菌落的频率为每70个G418抗性菌落中有1个。在使用相同靶向构建体的平行实验中，在每5500个未选择的菌落中发现1个hprt靶向细胞。因此，与未选择的菌落群体相比，在用共导入DNA转化的菌落群体中观察到靶向细胞富集了80倍。共转化后靶向细胞的这种富集在分离含有靶向但不可选择基因改变的菌落时应该是有用的。
