Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells-猫眼课题宝

胚胎干细胞中插入和替换载体的目标频率和整合模式

DOI：

10.1128/mcb.11.9.4509-4517.1991

发表时间：

1991

期刊：

Molecular and Cellular Biology

影响因子：

5.3

通讯作者：

中科院分区：

生物学2区

文献类型：

作者：

Paul Hasty;J. Rivera;Christine Chang;Allan Bradley

基因靶向已被用于引导突变进入小鼠胚胎干细胞（ES）的特定染色体位点。改变的位点可以在嵌合体体内进行研究，如果突变的细胞对生殖系有贡献，则可以在其后代中进行研究。虽然同源重组是广泛应用的基因靶向技术的基础，但迄今为止，在哺乳动物细胞中，载体与染色体靶标之间的同源重组机制基本上是未知的。在这里，我们通过比较插入载体和靶向hprt的替代载体来研究胚胎干细胞中基因靶向的本质。我们发现插入载体的靶向频率比具有相同同源序列长度的替代载体高9倍。我们还观察到，大多数用替代载体靶向的克隆没有像预测的那样重组。重组结构分析表明，外源异源序列经常被纳入目标位点。这种观察结果可以通过再循环载体的单次反向重组（载体插入）或载体串联体的双次反向重组/基因转换（基因替换）来解释。因此，插入矢量的单次反向重组比在长臂和短臂上具有交叉连接的替代矢量的双次反向重组发生的频率高92倍。

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.