摘要
[目的]利用CRISPR/Cas9技术对猪内源性逆转录病毒(PERV)进行编码区的大片段敲除,获得PERV敲除的阳性PK15单克隆细胞系。[方法]通过对巴马小型猪基因组进行高通量测序,获得PERV高度保守序列,再根据CRISPR/Cas9的构建原理,设计2个gRNA,并将其同时整合入含有PB转座子的可表达Cas9蛋白的载体中,即PB-CAG-2×gRNA-Cas9载体。然后以电转方式将打靶载体转入PK15细胞中,并通过药物(G418)筛选方法筛出单细胞克隆。通过PCR鉴定,挑出阳性克隆,并将其传代扩大获得细胞系。[结果]酶切、测序验证了载体PB-CAG-2×gRNA-Cas9的正确性,PCR结果显示7株单克隆细胞系均有约3600bp的PERV大片段敲除。[结论]构建了PERV敲除的基因打靶载体PB-CAG-2×gRNA-Cas9,并利用该载体成功打靶获得了7株PERV大片段敲除的PK15细胞系。
[Objective]To establish monoclonal PK15 cell lines with long fragments deletions of porcine endogenous retrovirus(PERV)by CRISPR/Cas9.[Methods]Through high-throughput sequencing of the Bama small pig genome,the highly conserved sequence of PERV was obtained,and according to the guideline of construction of CRISPR/Cas9,2 gRNAs were designed and integrated into the Cas9 expressing vector containing with PB Transposon,that is,the PB-CAG-2×gRNA-Cas9 vector.By means of electroporation of the targeting vector and screening by drug G418,single cell cloning were chosen and identified by PCR.[Results]According to the restriction enzymes digestion and sequencing analysis,PB-CAG-2×gRNA-Cas9 vector was successfully constructed.In addition,the PCR results showed all the 7 monoclonal cell lines had about 3600bp of PERV large fragment knockout.[Conclusion]The efficient gene targeting vector of PB-CAG-2×gRNA-Cas9 was constructed,and by the use of this vector,7 monoclonal PK15 cell lines with large fragment knockout of PERV were obtained.This study will provide a pathway for the construction of PERV knockout cloned pigs.
作者
蒙红毅
张健民
李黎
高毅
Hongyi Meng;Jianmin Zhang;Li Li;Yi Gao(Zhujiang Hospital,Southern Medical University,Guangzhou 510280,China)
出处
《生物技术》
CAS
2018年第6期513-519,525,共8页
Biotechnology
基金
国家重点研发计划项目(“基于hiHep/HepGL细胞和ZhJ-Ⅲ装置的混合型人工肝系统的构建与开发”,No.2018YFC1106400)
国家自然科学基金项目(“仿肝板结构肝组织三维培养回复并维持肝细胞极性的机制研究”,No.81470875
“体细胞核移植培育CRISPR/Cas9技术敲除PERV基因克隆猪的研究”,No.81701580)
广东省科技计划项目(“ZhJ-3型生物人工肝系统组织工程构建与转化研究”,No.2015B020229002)
广东省自然科学基金项目(“生物人工肝应用基础研究”,No.2014A030312013
“纤维支架循环灌注大规模培养系统激活Rac-Par3通路重建并维持肝细胞极性的机制研究”,No.2018A030313128
“抑制mTORC2信号诱导肝细胞癌自噬性死亡的机制研究”,No.2018A030313214)
广州市科技计划项目(“人肝细胞特异性支架材料的优化和开发”,No.201803010086)
作者简介
蒙红毅(1990-),女,广东省茂名人,医学硕士生,研究方向:异种器官移植,Email:menghongyi2018@163.com;通讯作者:高毅(1961-),男,博士,教授,博士/博士后导师,研究方向:生物人工肝、肝脏组织工程、肝移植,发表论文100余篇,被SCI、EI收录近40篇,申请专利41项,获授权19项,Email:gaoyi6146@163.com。
