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利用CRISPR/Cas9技术产生肌肉特异表达Cas9的小鼠胚胎 被引量:1

Generation of muscle-specific gene-targeted mouse embryos expressing Cas9 protein
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摘要 目的通过CRISPR/Cas9技术获得肌肉特异性表达Cas9蛋白小鼠胚胎,为建立肌肉特异表达Cas9小鼠动物模型奠定基础。方法设计小鼠Rosa26位点sgRNA并通过体外酶切验证活性,同时使用同源重组构建肌肉特异性同源打靶载体;通过显微注射将Rosa26sgRNA与Cas9蛋白注射到小鼠胚胎,通过PCR及测序检测胚胎的编辑情况,同时移植到假孕母鼠体内,待其生产;将同源打靶载体与Rosa26sgRNA和Cas9一起注射到小鼠胚胎,通过PCR检测整合情况。结果体外酶切实验表明,体外转录的sgRNA与Cas9蛋白联合可对靶位点产生编辑作用;成功构建了肌肉特异性同源打靶载体Donor-SP-px459;通过原核注射获得Rosa26基因编辑胚胎,经移植获得Rosa26基因编辑小鼠;注射同源打靶载体后,成功获得肌肉特异表达Cas9蛋白的基因打靶小鼠胚胎。结论利用CRISPR/Cas9技术,成功获得Rosa26基因编辑胚胎和小鼠,并获得了肌肉特异性表达Cas9蛋白小鼠胚胎,为利用基因打靶构建肌肉特异表达Cas9的小鼠动物模型奠定基础。 Objective To obtain gene-targeted mouse embryos expressing Cas9 protein specifically in muscle using CRISPR/Cas9,to lay the foundation for the construction of animal model expressing Cas9 protein specifically in muscle.Methods The sgRNA targeted Rosa26 locus was designed and synthesized according to the sequence of the Rosa26 gene in mice.The editing efficiency was tested by in vitro cleavage assay.Rosa26 sgRNA and Cas9 protein were subsequently co-injected into mouse pronuclear-stage embryos to examine the editing efficiency in vivo.A mouse Rosa26 locus homologous targeting vector for the muscle-specific expression of Cas9 protein was designed and constructed.Finally,gene-targeting embryos were obtained through co-injecting the homologous targeting vector and Rosa26 sgRNA with Cas9 protein into mouse embryos.Results The sgRNA designed and synthesized according to the sequence of mouse Rosa26 gene was effective at gene editing in the in vitro digestion experiments.The homologous targeting vector for the muscle-specific expression of Cas9 protein,Donor-SP-px459,was successfully constructed by PCR and homologous recombination ligation.Mouse embryos injected with Rosa26 sgRNA and Cas9 protein showed normal cleavage and supported blastocyst development.PCR and sequencing result demonstrated that gene editing occurred in the obtained embryos,after which the embryo grew into a Rosa26 gene edited mouse.Mouse embryos for the muscle-specific expression of Cas9 protein were successfully obtained after co-injection of a homologous targeting vector and Rosa26 sgRNA with Cas9 protein.Conclusions Rosa26 gene-edited embryos and mice are obtained using the CRISPR/Cas9 system,and mouse embryos with muscle-specific expression of Cas9 protein are successfully obtained,laying the foundation for the construction of animal models expressing Cas9 protein specifically in muscle.
作者 李昊 陈胜男 李松美 朴善花 安铁洙 王春生 LI Hao;CHEN Shengnan;Li Songmei;PIAO Shanhua;AN Tiezhu;WANG Chunsheng(Laboratory of Developmental Biology,College of Life Science,Northeast Forestry University,Harbin 150040,China)
出处 《中国实验动物学报》 CAS CSCD 北大核心 2019年第3期271-277,共7页 Acta Laboratorium Animalis Scientia Sinica
基金 中央高校基本科研业务费专项C类项目资金资助(2572016CA10) 黑龙江省自然科学基金项目(C2016012)~~
关键词 Rosa26 Cas9 基因编辑 肌肉特异 基因打靶 Rosa26 Cas9 gene editing muscle-specific gene targeting
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