Generating mutant rats using the Sleeping Beauty transposon system
Generating mutant rats using the Sleeping Beauty transposon system
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DOI：
10.1016/j.ymeth.2009.04.010
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发表时间：
2009-11-01
期刊：
影响因子：
4.8
通讯作者：
Horie, Kyoji
中科院分区：
文献类型：
--
作者：
Kitada, Kazuhiro;Keng, Vincent W.;Horie, Kyoji
实验室大鼠是生物医学研究的宝贵动物模型。然而，突变体大鼠资源仍然有限，需要开发大规模产生突变体的方法。我们最近利用睡美人（SB）转座子系统开发了一种快速产生插入突变大鼠的方法。首先，产生携带单一转基因的转基因大鼠，即SB转座子载体和SB转座酶。其次，将这些单转基因大鼠杂交以获得携带两种转基因的双转基因大鼠。 SB转座子被调动到这些双转基因大鼠的种系中，重新插入基因组中的另一个位置，并在后代中获得杂合突变大鼠。通过转座子载体中整合的绿色荧光蛋白 (GFP) 报告基因，利用 PolyA-trap 方法快速、非侵入性地识别基因插入事件。突变基因通过接头连接介导的 PCR 或 cDNA 末端的 3' 快速扩增 (TRACE) 进行确认。突变基因的内源表达谱也可以使用作为转座子载体中的启动子捕获单元并入的 LacZ 基因来可视化。该方法简单易行，易于适用于其他转座子系统，并且将成为生物医学研究界宝贵的突变大鼠资源。 (C) 2009 Elsevier Inc. 保留所有权利。
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 (TRACE). 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. (C) 2009 Elsevier Inc. All rights reserved.