摘要
目的家兔是一种重要的模式动物,然而目前家兔中缺乏外源基因定点整合的相关技术。方法为了在兔胚胎成纤维细胞水平建立起成熟的H11位点定点整合平台,试验根据文献报道的人和小鼠的H11位点鉴定出家兔的H11位点,并使用CRISPR/Cas9系统和同源重组技术,设计了两对针对H11位点的sgRNA和左右同源臂,构建敲除载体和打靶载体。将两种载体共同转染进兔胚胎成纤维细胞中,在共转染的细胞基因组中依托嘌呤霉素筛选检测外源基因整合。结果在共转染的兔胚胎成纤维细胞中检测到外源基因插入,并且在敲入后成功表达外源基因。结论该实验依赖于CRISPR/Cas9技术和同源重组技术在细胞水平上创建高效的位点特异性整合系统,为未来转基因兔的安全和有效制备奠定了基础。
Objective Rabbits are important model animals,However,there is short of technology for site-specific integration of exogenous genes at present in rabbits.Methods In order to establish a mature site-specific integration platform for H11 site at the level of rabbits’fibroblasts,this experiment identified the H11 site of rabbits based on human and mouse H11 site reported in the literature.Meanwhile,two pairs of sgRNAs and left and right homology arms targeting the H11 site were designed by using the CRISPR/Cas9 system and homologous recombination technology,and constructed the knockout vectors and the targeting vectors.The two vectors were co-transfected into rabbit embryonic fibroblasts,and exogenous gene integration was detected by puromycin selection in the co-transfected cell genome.Results showed that exogenous gene insertion was detected in co-transfected rabbit embryonic fibroblasts,and the foreign gene was successfully expressed after knock-in.Conclusion This experiment relies on that CRISPR/Cas9 technology and homologous recombination technology to create a highly efficient site-specific integration system at the cellular level,laying the foundation for the safe and efficient preparation of future transgenic rabbits.
作者
姚洋
尤双
刘芝瑾
张翔宇
侯晓旭
郭涛
倪伟
胡圣伟
YAO Yang;YOU Shuang;LIU Zhijin;ZHANG Xiangyu;HOU Xiaoxu;GUO Tao;NI Wei;HU Shengwei(College of Life Science,Shihezi University,Shihezi,Xinjiang 832003,China)
出处
《石河子大学学报（自然科学版）》
CAS
北大核心
2019年第5期580-588,共9页
Journal of Shihezi University(Natural Science)
基金
新疆生产建设兵团科技创新中青年领军人才项目(2016BC001,2017CB003)
石河子大学青年科技创新人才项目(CXRC201603)
