Suppression of nonsense-mediated mRNA decay permits unbiased gene trapping in mouse embryonic stem cells.
Suppression of nonsense-mediated mRNA decay permits unbiased gene trapping in mouse embryonic stem cells.
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DOI：
10.1093/nar/gni022
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发表时间：
2005-02-01
影响因子：
14.9
通讯作者：
Ishida Y
中科院分区：
文献类型：
--
作者：
Shigeoka T;Kawaichi M;Ishida Y
一个国际合作项目已经提出，使用随机和靶向插入诱变技术的组合，在胚胎干细胞（ES）中克隆所有小鼠基因。随机基因捕获将是初始阶段的首选，然后将进行基因靶向实验，以单独敲除剩余的“难以捕获”基因。随机插入诱变最受欢迎的技术之一是启动子捕获，其仅破坏活跃转录的基因。另一方面，聚腺苷酸化（poly-A）捕获可以捕获更广泛的基因，包括那些在靶细胞中不表达的基因，但我们注意到，它不可避免地选择载体整合到被捕获基因的最后一个内含子中。在这里，我们提出的证据表明，这种显着的倾斜是由用于多聚腺苷酸捕获通过mRNA监视机制，无义介导的mRNA衰变（NMD）的选择性标记mRNA的降解。我们还报告了一种新的多聚-A-陷阱策略，UPATrap，它抑制NMD的选择性标记mRNA，并允许捕获的转录沉默的基因，而没有偏见的载体整合位点的发展。我们相信UPATrap技术能够简单而直接地无偏地灭活ES细胞中的所有小鼠基因。
An international collaborative project has been proposed to inactivate all mouse genes in embryonic stem (ES) cells using a combination of random and targeted insertional mutagenesis techniques. Random gene trapping will be the first choice in the initial phase, and gene-targeting experiments will then be carried out to individually knockout the remaining ‘difficult-to-trap’ genes. One of the most favored techniques of random insertional mutagenesis is promoter trapping, which only disrupts actively transcribed genes. Polyadenylation (poly-A) trapping, on the other hand, can capture a broader spectrum of genes including those not expressed in the target cells, but we noticed that it inevitably selects for the vector integration into the last introns of the trapped genes. Here, we present evidence that this remarkable skewing is caused by the degradation of a selectable-marker mRNA used for poly-A trapping via an mRNA-surveillance mechanism, nonsense-mediated mRNA decay (NMD). We also report the development of a novel poly-A-trap strategy, UPATrap, which suppresses NMD of the selectable-marker mRNA and permits the trapping of transcriptionally silent genes without a bias in the vector-integration site. We believe the UPATrap technology enables a simple and straightforward approach to the unbiased inactivation of all mouse genes in ES cells.
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影响因子：
64.8
作者：
Zambrowicz, BP;Friedrich, GA;Sands, AT
通讯作者：
Sands, AT
DOI：
10.1073/pnas.1633296100
发表时间：
2003-08-19
影响因子：
11.1
作者：
Hansen, J;Floss, T;Ruiz, P
通讯作者：
Ruiz, P
影响因子：
9.2
作者：
Dostie, J;Dreyfuss, G
通讯作者：
Dreyfuss, G
影响因子：
2.5
作者：
You, Y;Bersgtram, R;Schimenti, J
通讯作者：
Schimenti, J
影响因子：
16
作者：
Ohnishi, T;Yamashita, A;Ohno, S
通讯作者：
Ohno, S