Deng C, Thomas K R, Capecchi M R
Howard Hughes Medical Institute, Eccles Institute of Human Genetics, University of Utah School of Medicine, Salt Lake City 84112.
Mol Cell Biol. 1993 Apr;13(4):2134-40. doi: 10.1128/mcb.13.4.2134-2140.1993.
Gene targeting was used to introduce nonselectable genetic changes into chromosomal loci in mouse embryo-derived stem cells. The nonselectable markers were linked to a selectable marker in both insertion- and replacement-type vectors, and the transfer of the two elements to the Hprt locus was assayed. When insertion vectors were used as substrates, the frequency of transfer was highly dependent upon the distance between the nonselectable marker and the double-strand break in the vector. A marker located close to the vector ends was frequently lost, suggesting that a double-strand gap repair activity is involved in vector integration. When replacement vectors were used, cotransfer of a selectable marker and a nonselectable marker 3 kb apart was over 50%, suggesting that recombination between vector and target often occurs near the ends of the vector. To illustrate the use of replacement vectors to transfer specific mutations to the genome, we describe targeting of the delta F508 mutation to the CFTR gene in mouse embryo-derived stem cells.
基因打靶被用于将不可选择的遗传变化引入小鼠胚胎干细胞的染色体位点。在插入型和置换型载体中，不可选择标记均与一个可选择标记相连，并检测这两个元件向次黄嘌呤磷酸核糖转移酶（Hprt）位点的转移情况。当使用插入型载体作为底物时，转移频率高度依赖于不可选择标记与载体中双链断裂之间的距离。靠近载体末端的标记经常丢失，这表明双链缺口修复活性参与了载体整合。当使用置换型载体时，相距3 kb的一个可选择标记和一个不可选择标记的共转移率超过50%，这表明载体与靶标之间的重组经常发生在载体末端附近。为了说明使用置换型载体将特定突变转移到基因组中的方法，我们描述了将ΔF508突变靶向小鼠胚胎干细胞中的囊性纤维化跨膜传导调节因子（CFTR）基因的过程。
