Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection.
Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection.
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DOI：
10.1186/s13104-015-1241-6
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发表时间：
2015-06-30
影响因子：
1.8
通讯作者：
Adachi N
中科院分区：
文献类型：
--
作者：
Saito S;Ura K;Kodama M;Adachi N
同源重组的靶向基因修饰为研究细胞和动物的基因功能提供了有力的工具。在高等真核生物中，靶向载体的非同源整合比靶向整合发生的频率要高几个数量级，这使得基因靶向技术效率极低。由于这个原因，负选择策略已被用于减少与非同源载体整合相关的耐药克隆的数量，特别是当在目标位点引入DNA断裂的人工核酸酶不可用或不需要时。因此，使用无启动子的耐药标记基因的外显子陷阱策略提供了反选择非同源整合子的有效方法。然而，构建外显子捕获靶向载体是一个耗时且复杂的过程。通过高效的at介导重组，我们成功地开发了一种简单快速的方法来构建基于质粒的载体，允许外显子捕获基因靶向。这些外显子诱捕载体可用于在小鼠胚胎干细胞和人HT1080细胞中获得正确的靶向克隆。最重要的是，通过使用一个条件细胞毒性基因，我们进一步开发了一种新的负选择策略，从而提高了外显子陷阱载体非同源整合的反选择效率。我们的方法将极大地促进外显子捕获基因靶向技术在哺乳动物细胞，特别是当与新的负选择策略相结合。本文的在线版本（doi:10.1186/s13104-015-1241-6）包含补充材料，可供授权用户使用。
Targeted gene modification by homologous recombination provides a powerful tool for studying gene function in cells and animals. In higher eukaryotes, non-homologous integration of targeting vectors occurs several orders of magnitude more frequently than does targeted integration, making the gene-targeting technology highly inefficient. For this reason, negative-selection strategies have been employed to reduce the number of drug-resistant clones associated with non-homologous vector integration, particularly when artificial nucleases to introduce a DNA break at the target site are unavailable or undesirable. As such, an exon-trap strategy using a promoterless drug-resistance marker gene provides an effective way to counterselect non-homologous integrants. However, constructing exon-trapping targeting vectors has been a time-consuming and complicated process. By virtue of highly efficient att-mediated recombination, we successfully developed a simple and rapid method to construct plasmid-based vectors that allow for exon-trapping gene targeting. These exon-trap vectors were useful in obtaining correctly targeted clones in mouse embryonic stem cells and human HT1080 cells. Most importantly, with the use of a conditionally cytotoxic gene, we further developed a novel strategy for negative selection, thereby enhancing the efficiency of counterselection for non-homologous integration of exon-trap vectors. Our methods will greatly facilitate exon-trapping gene-targeting technologies in mammalian cells, particularly when combined with the novel negative selection strategy. The online version of this article (doi:10.1186/s13104-015-1241-6) contains supplementary material, which is available to authorized users.