Efficient transposition of the Tol2 transposable element from a single-copy donor in zebrafish
Efficient transposition of the Tol2 transposable element from a single-copy donor in zebrafish
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DOI：
10.1073/pnas.0810380105
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发表时间：
2008-12-16
影响因子：
11.1
通讯作者：
Kawakami, Koichi
中科院分区：
文献类型：
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作者：
Urasaki, Akihiro;Asakawa, Kazuhide;Kawakami, Koichi
Tol2转座因子是模式脊椎动物中的一种强大的遗传工具，已被用于转基因、插入诱变、基因捕获和增强子捕获。然而，尚未开发使用Tol 2的体内转座系统。在这里，我们报告了在体内Tol2转座系统的模式脊椎动物，斑马鱼。首先，我们构建了转基因斑马鱼，其基因组上携带Tol2的单拷贝整合，并将转座酶mRNA注射到单细胞期胚胎中。Tol2插入在生殖谱系中被有效地动员。然后，我们动员了插入的Tol2基因陷阱构建的nup214基因，这导致了隐性致死突变表型，并证明了这种方法是适用于从转座子插入突变体的回复突变体的分离。第二，我们构建了转基因鱼携带的转座酶的cDNA下的热休克蛋白70启动子的控制。将含有转座酶基因和单拷贝Tol2插入的双转基因鱼在成体阶段进行热休克处理。我们发现转座可以在雄性生殖细胞中有效地诱导。我们分析了新的整合位点，发现它们中的大多数（83%）被映射到转座子供体染色体以外的染色体上，9%的本地跳跃事件映射到距离供体基因座不到300 kb的地方。我们目前的研究表明，在体内Tol2转座系统是有用的创建全基因组插入单拷贝供体，并应促进脊椎动物的功能基因组学和转座子生物学。
The Tol2 transposable element is a powerful genetic tool in model vertebrates and has been used for transgenesis, insertional mutagenesis, gene trapping, and enhancer trapping. However, an in vivo transposition system using Tol2 has not yet been developed. Here we report the in vivo Tol2 transposition system in a model vertebrate, zebrafish. First, we constructed transgenic zebrafish that carried single-copy integrations of Tol2 on the genome and injected transposase mRNA into one-cell stage embryos. The Tol2 insertions were mobilized efficiently in the germ lineage. We then mobilized an insertion of the Tol2 gene trap construct in the nup214 gene, which caused a recessive lethal mutant phenotype, and demonstrated that this method is applicable to the isolation of revertants from a transposon insertional mutant. Second, we constructed transgenic fish carrying the transposase cDNA under the control of the hsp70 promoter. Double-transgenic fish containing the transposase gene and a single-copy Tol2 insertion were treated with heat shock at the adult stage. We found that transposition can be induced efficiently in the male germ cells. We analyzed new integration sites and found that the majority (83%) of them were mapped on chromosomes other than the transposon donor chromosomes and that 9% of local hopping events mapped less than 300 kb away from the donor loci. Our present study demonstrates that the in vivo Tol2 transposition system is useful for creating genome-wide insertions from a single-copy donor and should facilitate functional genomics and transposon biology in vertebrates.