Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells
Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells
复制标题
DOI：
10.1038/nprot.2011.378
复制
发表时间：
2011-10-01
期刊：
影响因子：
14.8
通讯作者：
Hamra, F. Kent
中科院分区：
文献类型：
--
作者：
Ivics, Zoltan;Izsvak, Zsuzsanna;Hamra, F. Kent
我们描述了一种利用睡美人(SB)转座子介导的插入突变在大鼠精原干细胞(SSCs)中产生突变等位基因的实验方法。该方案是基于将诱变基因陷阱转座子从转基因的质粒载体动员到培养干细胞的基因组中。在表达基因中插入转座子的细胞是基于由转座子编码的抗生素抗性基因的激活而选择的。这些基因陷阱克隆被移植到受体雄性的睾丸中(作为单克隆或多克隆文库)；这些创始人与野生型雌性杂交后，插入的片段就可以传递给F-1后代。这一简单、经济和用户友好的方法管道使筛选大鼠的功能基因注释成为可能，并适用于其他已建立有生殖系能力的干细胞的脊椎动物模型。从SSCs的转染到含有基因组SB基因陷阱插入的杂合子F-1后代的基因分型需要5-6个月的完整程序。
We describe an experimental approach for generating mutant alleles in rat spermatogonial stem cells (SSCs) using Sleeping Beauty (SB) transposon-mediated insertional mutagenesis. The protocol is based on mobilization of mutagenic gene-trap transposons from transfected plasmid vectors into the genomes of cultured stem cells. Cells with transposon insertions in expressed genes are selected on the basis of activation of an antibiotic-resistance gene encoded by the transposon. These gene-trap clones are transplanted into the testes of recipient males (either as monoclonal or polyclonal libraries); crossing of these founders with wild-type females allows the insertions to be passed to F-1 progeny. This simple, economic and user-friendly methodological pipeline enables screens for functional gene annotation in the rat, with applicability in other vertebrate models where germ line-competent stem cells have been established. The complete protocol from transfection of SSCs to the genotyping of heterozygous F-1 offspring that harbor genomic SB gene-trap insertions takes 5-6 months.