Haploid mouse embryonic stem cells (ESCs), in which a single hit mutation is sufficient to produce loss-of-function phenotypes, have provided a powerful tool for forward genetic screening. This strategy, however, can be hampered by undesired autodiploidization of haploid ESCs. To overcome this obstacle, we designed a new methodology that facilitates enrichment of homozygous mutant ESC clones arising from autodiploidization during haploid gene trap mutagenesis. Haploid mouse ESCs were purified by fluorescence-activated cell sorting to maintain their haploid property and then transfected with the Tol2 transposon-based biallelically polyA-trapping (BPATrap) vector that carries an invertible G418 plus puromycin double selection cassette. G418 plus puromycin double selection enriched biallelic mutant clones that had undergone autodiploidization following a single vector insertion into the haploid genome. Using this method, we successfully generated 222 homozygous mutant ESCs from 2208 clones by excluding heterozygous ESCs and ESCs with multiple vector insertions. This relatively low efficiency of generating homozygous mutant ESCs was partially overcome by cell sorting of haploid ESCs after Tol2 BPATrap transfection. These results demonstrate the feasibility of our approach to provide an efficient platform for mutagenesis of ESCs and functional analysis of the mammalian genome.

中文翻译：

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单倍体基因陷阱诱变过程中自二倍体化引起的纯合突变小鼠胚胎干细胞的收集

单倍命中突变足以产生功能丧失表型的单倍体小鼠胚胎干细胞（ESC），为正向遗传筛选提供了强大的工具。但是，单倍体ESC的不希望的二倍体化会阻碍这种策略。为了克服这一障碍，我们设计了一种新方法，可促进单倍体基因陷阱诱变过程中自二倍体化引起的纯合突变ESC克隆的富集。通过荧光激活细胞分选纯化单倍体小鼠ESC，以保持其单倍体特性，然后用Tol2转染基于转座子的双烯丙基聚A捕获（BPATrap）载体，该载体带有可逆G418加嘌呤霉素双选择盒。G418加嘌呤霉素的双重选择富集了双等位基因突变体克隆，这些克隆在单载体插入单倍体基因组后就经历了自二倍体化。使用此方法，我们通过排除杂合子ESC和具有多个载体插入的ESC，成功地从2208个克隆中生成了222个纯合突变体ESC。在Tol2 BPATrap转染后，通过单倍体ESC的细胞分选，部分克服了产生纯合突变ESC的这种相对较低的效率。这些结果证明了我们的方法为ESC的诱变和哺乳动物基因组功能分析提供有效平台的可行性。