Di Domenico Alexandra I, Christodoulou Ioannis, Pells Steve C, McWhir Jim, Thomson Alison J
Division of Gene Function and Development, Roslin Institute (Edinburgh), Roslin, EH25 9PS, UK.
Cloning Stem Cells. 2008 Jun;10(2):217-30. doi: 10.1089/clo.2008.0016.
Genetic modification of human embryonic stem cells (hESCs) will be an essential tool to allow full exploitation of these cells in regenerative medicine and in the study of hESC biology. Here we report multiple sequential modifications of an endogenous gene (hprt) in hESCs. A selectable marker flanked by heterospecific lox sites was first introduced by homologous recombination (HR) into the hprt gene. In a subsequent step, exchange of the selectable marker with another cassette was achieved by recombinase-mediated cassette exchange (RMCE). We show that 100% of the recovered clones were the result of RMCE using a promoter trap strategy at the hprt locus. hprt-targeted H1 cells maintained a diploid karyotype and expressed hESC surface markers before and after RMCE. Finally, we report a double replacement strategy using two sequential gene targeting steps resulting in the targeted correction of an hprt-mutated hESC line.
对人类胚胎干细胞（hESCs）进行基因改造，将成为在再生医学和hESC生物学研究中充分利用这些细胞的重要工具。在此，我们报告了hESCs中一个内源性基因（hprt）的多次连续改造。首先通过同源重组（HR）将一个两侧带有异源lox位点的选择标记引入hprt基因。在随后的步骤中，通过重组酶介导的盒式交换（RMCE）实现了选择标记与另一个盒式结构的交换。我们表明，使用hprt基因座处的启动子捕获策略，100%的回收克隆是RMCE的结果。靶向hprt的H1细胞在RMCE前后均保持二倍体核型并表达hESC表面标志物。最后，我们报告了一种使用两个连续基因靶向步骤的双替换策略，该策略可对hprt突变的hESC系进行靶向校正。
