Generating mutant rats using the Sleeping Beauty

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• 作者：Kazuhiro Kitada ; Vincent W. Keng ; Junji Takeda ; Kyoji Horie
• 关键词：Mutagenesis ; Transposon ; Sleeping Beauty ; Rat
• 刊名：Methods
• 出版年：2009
• 出版时间：November 2009
• 年：2009
• 卷：49
• 期：3
• 页码：236-242
• 全文大小：280 K

文摘

The laboratory rat is an invaluable animal model for biomedical research. However, mutant rat resource is still limited, and development of methods for large-scale generation of mutants is anticipated. We recently utilized the Sleeping Beauty (SB) transposon system to develop a rapid method for generating insertional mutant rats. Firstly, transgenic rats carrying single transgenes, namely the SB transposon vector and SB transposase, were generated. Secondly, these single transgenic rats were interbred to obtain doubly-transgenic rats carrying both transgenes. The SB transposon was mobilized in the germline of these doubly-transgenic rats, reinserted into another location in the genome and heterozygous mutant rats were obtained in the progeny. Gene insertion events were rapidly and non-invasively identified by the green fluorescence protein (GFP) reporter incorporated in the transposon vector, which utilizes a polyA-trap approach. Mutated genes were confirmed by either linker ligation-mediated PCR or 3′-rapid amplification of cDNA ends (3′RACE). Endogenous expression profile of the mutated gene can also be visualized using the LacZ gene incorporated as a promoter-trap unit in the transposon vector. This method is straightforward, readily applicable to other transposon systems, and will be a valuable mutant rat resource to the biomedical research community.