Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons
Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons
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DOI：
10.1038/nprot.2014.008
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发表时间：
2014-04-01
期刊：
影响因子：
14.8
通讯作者：
Izsvak, Zsuzsanna
中科院分区：
文献类型：
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作者：
Ivics, Zoltan;Mates, Lajos;Izsvak, Zsuzsanna
我们描述了一个高效率的生殖系转基因和持续的转基因表达在两个重要的生物医学模型，小鼠和大鼠，通过使用睡美人转座子系统的协议。该方法是基于将编码SB100X超活性转座酶的合成mRNA与携带转基因构建体的环状质粒DNA一起共注射到受精卵母细胞的原核中，所述转基因构建体两侧是转座酶的结合位点。在转座酶mRNA翻译后，酶介导的转基因盒从注射的质粒中切除，随后进行永久基因组插入，产生稳定的转基因动物。通过使用该方案产生生殖系转基因创始动物需要近似3个月。转座子介导的转基因在效率和可靠的转基因表达方面与经典的原核显微注射相比是有利的，并且它提供了与慢病毒方法相当的功效，而不限制载体设计、转基因沉默问题以及与病毒载体一起工作的毒性和生物安全性问题。
We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in two important biomedical models, the mouse and the rat, by using the Sleeping Beauty transposon system. The procedure is based on co-injection of synthetic mRNA encoding the SB100X hyperactive transposase, together with circular plasmid DNA carrying a transgene construct flanked by binding sites for the transposase, into the pronuclei of fertilized oocytes. Upon translation of the transposase mRNA, enzyme-mediated excision of the transgene cassettes from the injected plasmids followed by permanent genomic insertion produces stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes similar to 3 months. Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression with classic pronuclear microinjection, and it offers comparable efficacies to lentiviral approaches without limitations on vector design, issues of transgene silencing, and the toxicity and biosafety concerns of working with viral vectors.