Random gene trapping is the application of insertional mutagenesis techniques that are conventionally used to inactivate protein‐coding genes in mouse embryonic stem (ES) cells. Transcriptionally silent genes are not effectively targeted by conventional random gene trapping techniques, thus we herein developed an unbiased poly (A) trap (UPATrap) method using a Tol2 transposon, which preferentially integrated into active genes rather than silent genes in ES cells. To achieve efficient trapping at transcriptionally silent genes using random insertional mutagenesis in ES cells, we generated a new diphtheria toxin (DT)‐mediated trapping vector, DTrap that removed cells, through the expression of DT that was induced by the promoter activity of the trapped genes, and selected trapped clones using the neomycin‐resistance gene of the vector. We found that a double‐DT, the dDT vector, dominantly induced the disruption of silent genes, but not active genes, and showed more stable integration in ES cells than the UPATrap vector. The dDT vector disrupted differentiated cell lineage genes, which were silent in ES cells, and labeled trapped clone cells by the expression of EGFP upon differentiation. Thus, the dDT vector provides a systematic approach to disrupt silent genes and examine the cellular functions of trapped genes in the differentiation of target cells and development.

中文翻译：

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白喉毒素介导的转座子驱动的聚 (A) 捕获有效地破坏了胚胎干细胞中的转录沉默基因。

随机基因捕获是插入诱变技术的应用，该技术通常用于灭活小鼠胚胎干 (ES) 细胞中的蛋白质编码基因。传统的随机基因捕获技术无法有效靶向转录沉默基因，因此我们在此开发了一种使用Tol2的无偏聚 (A) 捕获 (UPATrap) 方法转座子，它优先整合到 ES 细胞中的活性基因而不是沉默基因中。为了在 ES 细胞中使用随机插入诱变实现对转录沉默基因的有效捕获，我们生成了一种新的白喉毒素 (DT) 介导的捕获载体，DTrap 去除细胞，通过被捕获的启动子活性诱导的 DT 表达。基因，并使用载体的新霉素抗性基因选择捕获的克隆。我们发现双 DT，即 dDT 载体，主要诱导沉默基因的破坏，而不是活性基因的破坏，并且在 ES 细胞中显示出比 UPATrap 载体更稳定的整合。dDT 载体破坏了在 ES 细胞中沉默的分化细胞谱系基因，并通过分化时 EGFP 的表达标记了被捕获的克隆细胞。因此，