Characterization of an exchangeable gene trap using pU‐17 carrying a stop codon‐βgeo cassette-猫眼课题宝

DOI：

10.1111/j.1440-169x.2005.00792.x

发表时间：

2005-04

期刊：

Development

影响因子：

4.6

通讯作者：

中科院分区：

生物学2区

文献类型：

作者：

Takuya Taniwaki;Kyoko Haruna;Hiroshi Nakamura;T. Sekimoto;Y. Oike;Takashi Imaizumi;Fumiyo Saito;Mayumi Muta;Yumi Soejima;Ayako Utoh;N. Nakagata;M. Araki;K. Yamamura;K. Araki

我们开发了一种新的可交换基因陷阱载体pU-17，携带内含子-lox 71-剪接受体（SA）-βgeo-loxP-pA-lox 2272-pSP 73-lox 511。SA含有三个终止密码子，与β半乳糖苷酶/新霉素抗性融合基因（βgeo）的ATG同框，可在启动子捕获中发挥作用。我们发现，陷阱载体是高度选择性的整合在内含子相邻的外显子含有起始密码子。此外，通过使用Cre‐mutant lox系统，我们成功地用增强型绿色荧光蛋白（EGFP）基因替换了βgeo基因，用替换的克隆建立了小鼠品系，通过与Flp‐deleter小鼠交配去除了选择标记基因，并确认了替换的EGFP基因以与βgeo基因相同的模式表达。因此，使用这种pU-17陷阱载体，我们可以首先进行随机诱变，然后通过Cre介导的重组，将βgeo基因替换为在陷阱启动子控制下表达的任何目的基因，将其转化为功能获得突变。

We have developed a new exchangeable gene trap vector, pU‐17, carrying the intron‐lox71‐splicing acceptor (SA)‐βgeo‐loxP‐pA‐lox2272‐pSP73‐lox511. The SA contains three stop codons in‐frame with the ATG of βgalactosidase/neomycin‐resistance fusion gene (βgeo) that can function in promoter trapping. We found that the trap vector was highly selective for integrations in the introns adjacent to the exon containing the start codon. Furthermore, by using the Cre‐mutant lox system, we successfully replaced the βgeo gene with the enhanced green fluorescent protein (EGFP) gene, established mouse lines with the replaced clones, removed the selection marker gene by mating with Flp‐deleter mice, and confirmed that the replaced EGFP gene was expressed in the same pattern as the βgeo gene. Thus, using this pU‐17 trap vector, we can initially carry out random mutagenesis, and then convert it to a gain‐of‐function mutation by replacing the βgeo gene with any gene of interest to be expressed under the control of the trapped promoter through Cre‐mediated recombination.

Characterization of an exchangeable gene trap using pU‐17 carrying a stop codon‐βgeo cassette

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