Tagging genes with cassette-exchange sites
Tagging genes with cassette-exchange sites
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DOI：
10.1093/nar/gni045
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Cortese, R
中科院分区：
文献类型：
--
作者：
Cobellis, G;Nicolaus, G;Cortese, R
为了使转基因更灵活和可重复，我们开发了一种基于新型5'和3'''基因陷阱'载体的系统，该载体含有异源特异性Flp识别靶位点和相应的'交换'载体，允许将任何感兴趣的DNA序列插入到陷阱位点中。Flp重组酶介导的盒式交换被证明是非常有效的，在我们的系统中，即使在没有基因座特异性选择。使用我们的基因捕获载体构建ES细胞克隆文库的可行性进行了测试，并通过RACE-PCR和测序在ES细胞中电穿孔后表征了一千个插入位点。我们在转基因小鼠体内验证了该系统的两个陷阱位点，并证明了插入陷阱位点的报告转基因具有与内源基因相同的表达模式。我们相信，这一系统将有助于在体内研究基因功能和大规模产生的小鼠模型的人类疾病，不仅造成损失，但也获得功能等位基因。
In an effort to make transgenesis more flexible and reproducible, we developed a system based on novel 5' and 3' `gene trap' vectors containing hetero-specific Flp recognition target sites and the corresponding `exchange' vectors allowing the insertion of any DNA sequence of interest into the trapped locus. Flp-recombinase-mediated cassette exchange was demonstrated to be highly efficient in our system, even in the absence of locus-specific selection. The feasibility of constructing a library of ES cell clones using our gene trap vectors was tested and a thousand insertion sites were characterized, following electroporation in ES cells, by RACE-PCR and sequencing. We validated the system in vivo for two trapped loci in transgenic mice and demonstrated that the reporter transgenes inserted into the trapped loci have an expression pattern identical to the endogenous genes. We believe that this system will facilitate in vivo studies of gene function and large-scale generation of mouse models of human diseases, caused by not only loss but also gain of function alleles.