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首页> 外文期刊>Molecular and Cellular Biology >Investigation of coelectroporation as a method for introducing small mutations into embryonic stem cells.
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Investigation of coelectroporation as a method for introducing small mutations into embryonic stem cells.

机译：作为将小突变引入胚胎干细胞的方法，研究电共穿孔。

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We have investigated coelectroporation as a method for introducing minor genetic changes into specific genes in embryonic stem cells. A selectable marker (neo) and a targeting replacement vector designed to insert a 4-bp insertion into exon 3 of the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene were coelectroporated into embryonic stem cells and selected in G418 and 6-thioguanine (6-TG). HPRT-negative clones were obtained at a frequency of approximately 1 per 520 G418r clones. Southern analysis and the polymerase chain reaction were used to demonstrate that 3 of 36 of the 6-TG-resistant clones had the desired 4-bp insertion without any other disruption of the HPRT locus. Initial studies indicated that the other 33 6-TG-resistant clones probably resulted from the targeted integration of a concatemer containing both the targeting construct and the selectable neo gene.
机译：我们已经研究了coelectroporation作为一种将微小的遗传变化引入胚胎干细胞中特定基因的方法。将选择标记（neo）和旨在将4-bp插入小鼠次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（HPRT）基因的外显子3插入的靶向替代载体共电穿孔进入胚胎干细胞，并在G418和6-硫鸟嘌呤中进行选择（6- TG）。 HPRT阴性克隆的获得频率约为每520个G418r克隆1个。 Southern分析和聚合酶链反应用于证明6-TG抗性克隆中的36个中的3个具有所需的4-bp插入，而没有任何其他HPRT基因座的破坏。最初的研究表明，其他33个6-TG耐药克隆可能是由于同时含有靶向构建体和选择性neo基因的串联体的靶向整合而产生的。

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