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李昊,陈胜男,李松美,朴善花,安铁洙,王春生.利用CRISPR/ Cas9 技术产生肌肉特异表达Cas9 的小鼠胚胎[J].中国实验动物学报,2019,27(3):271~277.
利用CRISPR/ Cas9 技术产生肌肉特异表达Cas9 的小鼠胚胎
Generation of muscle-specific gene-targeted mouse embryos expressing Cas9 protein
投稿时间:2019-04-01  
DOI:10. 3969 / j.issn.1005-4847. 2019. 03. 001
中文关键词:  Rosa26  Cas9  基因编辑  肌肉特异  基因打靶
英文关键词:Rosa26  Cas9  gene editing  muscle-specific  gene targeting
基金项目:
作者单位E-mail
李昊 东北林业大学生命科学学院动物发育研究室,哈尔滨 150040 jayhaoli01@ 163.com 
陈胜男 东北林业大学生命科学学院动物发育研究室,哈尔滨 150040  
李松美 东北林业大学生命科学学院动物发育研究室,哈尔滨 150040  
朴善花 东北林业大学生命科学学院动物发育研究室,哈尔滨 150040  
安铁洙 东北林业大学生命科学学院动物发育研究室,哈尔滨 150040  
王春生 东北林业大学生命科学学院动物发育研究室,哈尔滨 150040 wangchunsheng79@ 163.com 
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中文摘要:
      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Rosa26 sgRNA 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Rosa26 sgRNA 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?
英文摘要:
      Objective To obtain gene-targeted mouse embryos expressing Cas9 protein specifically in muscleusing CRISPR/ Cas9, to lay the foundation for the construction of animal model expressing Cas9 protein specifically inmuscle. Methods The sgRNA targeted Rosa26 locus was designed and synthesized according to the sequence of theRosa26 gene in mice. The editing efficiency was tested by in vitro cleavage assay. Rosa26 sgRNA and Cas9 protein weresubsequently co-injected into mouse pronuclear-stage embryos to examine the editing efficiency in vivo. A mouse Rosa26locus 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 Cas9protein into mouse embryos. Results The sgRNA designed and synthesized according to the sequence of mouse Rosa26gene was effective at gene editing in the in vitro digestion experiments. The homologous targeting vector for the musclespecific expression of Cas9 protein, Donor-SP-px459, was successfully constructed by PCR and homologous recombinationligation. Mouse embryos injected with Rosa26 sgRNA and Cas9 protein showed normal cleavage and supported blastocystdevelopment. PCR and sequencing result demonstrated that gene editing occurred in the obtained embryos, after which theembryo grew into a Rosa26 gene edited mouse. Mouse embryos for the muscle-specific expression of Cas9 protein weresuccessfully 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 musclespecificexpression of Cas9 protein are successfully obtained, laying the foundation for the construction of animal models expressing Cas9 protein specifically in muscle.
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