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首页> 外文期刊>Molecular and Cellular Biology >Recycling selectable markers in mouse embryonic stem cells.
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Recycling selectable markers in mouse embryonic stem cells.

机译：回收小鼠胚胎干细胞中的选择标记。

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As a result of gene targeting, selectable markers are usually permanently introduced into the mammalian genome. Multiple gene targeting events in the same cell line can therefore exhaust the pool of markers available and limit subsequent manipulations or genetic analysis. In this study, we describe the combined use of homologous and CRE-loxP-mediated recombination to generate mouse embryonic stem cell lines carrying up to four targeted mutations and devoid of exogenous selectable markers. A cassette that contains both positive and negative selectable markers flanked by loxP sites, rendering it excisable by the CRE protein, was constructed. Homologous recombination and positive selection were used to disrupt the Rep-3 locus, a gene homologous to members of the mutS family of DNA mismatch repair genes. CRE-loxP-mediated recombination and negative selection were then used to recover clones in which the cassette had been excised. The remaining allele of Rep-3 was then subjected to a second round of targeting and excision with the same construct to generate homozygous, marker-free cell lines. Subsequently, both alleles of mMsh2, another mutS homolog, were disrupted in the same fashion to obtain cell lines homozygous for targeted mutations at both the Rep-3 and mMsh2 loci and devoid of selectable markers. Thus, embryonic stem cell lines obtained in this fashion are suitable for further manipulation and analysis involving the use of selectable markers.
机译：作为基因靶向的结果，通常将选择标记永久地引入哺乳动物基因组中。因此，同一细胞系中的多个基因靶向事件可能会耗尽可用标记库，并限制后续操作或遗传分析。在这项研究中，我们描述了同源和CRE-loxP介导的重组的结合使用，以产生携带多达四个靶向突变且没有外源选择标记的小鼠胚胎干细胞系。构建了一个包含正向和负向选择标记的盒，该标记位于loxP位点两侧，可被CRE蛋白激发。同源重组和阳性选择被用于破坏Rep-3基因座，该基因与DNA错配修复基因的mutS家族成员同源。然后，使用CRE-loxP介导的重组和阴性选择来回收其中已切除该盒的克隆。然后，使用相同的构建体对其余的Rep-3等位基因进行第​​二轮靶向和切除，以产生纯合的，无标记的细胞系。随后，另一种mutS同源物mMsh2的两个等位基因以相同的方式被破坏，以获得在Rep-3和mMsh2基因座上均针对靶突变纯合的细胞系，并且没有选择标记。因此，以这种方式获得的胚胎干细胞系适用于涉及选择标记的进一步操作和分析。

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