Enrichment and efficient screening of ES cells containing a targeted mutation: the use of DT-A gene with the polyadenylation signal as a negative selection maker
Enrichment and efficient screening of ES cells containing a targeted mutation: the use of DT-A gene with the polyadenylation signal as a negative selection maker
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DOI：
10.1023/a:1008914020843
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发表时间：
1999-06-01
影响因子：
3
通讯作者：
Miyazaki, J
中科院分区：
文献类型：
--
作者：
Yanagawa, Y;Kobayashi, T;Miyazaki, J
通过同源重组在胚胎干（ES）细胞中的基因靶向可以以非常低的频率发生。为了在筛选前富集同源重组体，通常使用负选择标记，如胸苷激酶（TK）和白喉毒素A片段（DT-A）。在本研究中，我们利用带有多聚腺苷酸化信号的DT-A基因开发了一个阴性选择标记，命名为DT-ApA。为了确定阴性选择的靶向效率的差异，我们为每种阴性选择构建了三种不同的靶向载体（第一种，在3'端的TK，第二种，在5'和3'端的TK< 2 X TK >，第三种，在同源序列的5'端的DT-ApA）。使用这些构建体的基因靶向实验清楚地表明，使用DT-ApA的阴性选择比使用TK进行同源重组更有效，并且使用DT-ApA的阴性选择与使用2 X TK的阴性选择一样有效。由于DT-ApA比2X TK更适合于构建基因打靶载体，因此DT-ApA是一种有效的阴性选择标记。使用LA-PCR与基因组DNA从ES细胞克隆促进简单的同源重组的检测，这表明与LA-PCR的筛选是相容的使用较长的同源序列的两个手臂在载体设计。我们的研究结果表明，使用DT-ApA的负选择与LA-PCR的筛选应用程序一起确保了高效和节省时间的同源重组筛选。
Gene targeting in embryonic stem (ES) cells via homologous recombination can occur at very low frequency. In order to enrich homologous recombinants before screening, a negative selection marker, such as thymidine kinase (TK) and diphtheria toxin A fragment (DT-A), has been commonly used. In this study, we developed a negative selection marker using DT-A gene with polyadenylation signal and it was designated DT-ApA. To determine the difference in targeting efficiency of the negative selections, we constructed three different targeting vectors for each negative selection (first, TK at the 3' end, second, TK at both the 5' and 3' ends < 2 X TK >, and third, DT-ApA at the 5' end of the homologous sequences). Gene targeting experiments using these constructs clearly showed that negative selection using DT-ApA was more efficient than that using TK for homologous recombination and that negative selection using DT-ApA was as efficient as that using 2 X TK. Considering the fact that the use of DT-ApA is more convenient for construction of targeting vectors than that of 2 X TK, DT-ApA is an efficient negative selection marker.In addition, we examined long and accurate PCR (LA-PCR) for screening gene targeted clones. The use of LA-PCR with genomic DNAs from ES cell clones facilitated simple detection of homologous recombinants, suggesting that the screening with LA-PCR is compatible with the use of longer homologous sequences of both arms in vector design. Our results indicate that the use of DT-ApA for negative selection together with the application of LA-PCR for screening ensures efficient and time-saving screening for homologous recombinants.