A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish
A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish
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DOI：
10.1016/j.devcel.2004.06.005
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发表时间：
2004-07-01
影响因子：
11.8
通讯作者：
Mishina, M
中科院分区：
文献类型：
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作者：
Kawakami, K;Takeda, H;Mishina, M
我们在这里报告了使用 Tol2 转座子系统在斑马鱼中开发一种新型基因捕获方法。首先，我们建立了一种高效的转基因方法，其中将含有Tol2转座子载体的质粒DNA和体外合成的转座酶mRNA共注射到单细胞阶段胚胎中。插入基因组中的转座子载体可以高频率传递给F1后代，并且可以在转基因鱼中重现由特定启动子调节的基因表达。然后，我们构建了一个包含剪接受体和 GFP 基因的基于转座子的基因捕获载体，进行了基因捕获的试点筛选，并获得了在时间和空间限制模式下表达 GFP 的鱼。我们证实内源转录物确实被插入捕获，并且插入可能干扰被捕获基因的表达。我们建议我们的基因陷阱方法应该促进脊椎动物发育和器官发生的研究。
We report here development of a novel gene trap method in zebrafish using the Tol2 transposon system. First, we established a highly efficient transgenesis method in which a plasmid DNA containing the Tol2 transposon vector and the transposase mRNA synthesized in vitro were coinjected into one-cell stage embryos. The transposon vector inserted in the genome could be transmitted to the F1 progeny at high frequencies, and regulated gene expression by a specific promoter could be recapitulated in transgenic fish. Then we constructed a transposon-based gene trap vector containing a splice acceptor and the GFP gene, performed a pilot screen for gene trapping, and obtained fish expressing GFP in temporally and spatially restricted patterns. We confirmed the endogenous transcripts were indeed trapped by the insertions, and the insertion could interfere with expression of the trapped gene. We propose our gene trap approach should facilitate studies of vertebrate development and organogenesis.