Transposon vectors for gene-trap insertional mutagenesis in vertebrates
Transposon vectors for gene-trap insertional mutagenesis in vertebrates
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DOI：
10.1002/gene.20049
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发表时间：
2004-08-01
期刊：
影响因子：
1.5
通讯作者：
Hackett, PB
中科院分区：
文献类型：
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作者：
Clark, KJ;Geurts, AM;Hackett, PB
大多数脊椎动物基因的功能仍然是未知或不确定的。插入突变为识别和理解这些基因的功能提供了一种方法。转座子已经成功地用于低等生物和植物的插入突变，但直到睡美人（SB）转座子系统的激活，尚无迹象表明在脊椎动物中有活性的基于dna的转座子。在脊椎动物中，研究者驱动的插入突变依赖于逆转录病毒插入或选择胚胎干细胞系中裸DNA的低频整合。我们将高度活跃的SB转座子与基因捕获技术结合起来，证明转座子捕获可以用于脊椎动物的插入突变筛选。在我们的研究中，大约四分之一的陷阱插入出现在转录单位中，这一比率与随机整合相称。我们发现，与荧光蛋白报告基因偶联的基因陷阱可用于检测活斑马鱼胚胎中特定细胞中活性基因的插入，支持我们的转座子陷阱用于脊椎动物的高通量功能基因组筛选。(C) 2004 Wiley-Liss, Inc。
The function of most vertebrate genes remains unknown or uncertain. Insertional mutagenesis offers one approach to identify and understand the function of these genes. Transposons have been used successfully in lower organisms and plants for insertional mutagenesis, but until activation of the Sleeping Beauty (SB) transposon system, there was no indication of active DNA-based transposons in vertebrates. Investigator-driven insertional mutagenesis in vertebrates has relied on retroviral insertions or selection of low-frequency integration of naked DNA in ES cell lines. We have combined the highly active SB transposon with gene-trapping technology to demonstrate that transposon traps can be used for insertional mutagenesis screens in vertebrates. In our studies about one-fourth of the trap insertions appear to be in transcriptional units, a rate that is commensurate with random integration. We show that gene-traps coupled to a fluorescent protein reporter gene can be used to detect insertions into genes active in specific cells of living zebrafish embryos, supporting use of our transposon traps for high-throughput functional genomic screens in vertebrates. (C) 2004 Wiley-Liss, Inc.