联系我们 加入收藏
 
年期检索 高级检索
33
2025年8月5日 星期二
  2016, Vol. 24 Issue (8): 1243-1250    
  专题:动物基因编辑技术 本期目录 | 过刊浏览 | 高级检索 |
CRISPR/Cas9介导的ASGR1基因敲除猪制备
李西睿1,冯冲1,龙川1,纪慧丽1,魏静1,石宁宁1,曾国敏2,蒋应弟3,潘登科1,田兴华4
1. 中国农业科学院北京畜牧兽医研究所
2. 甘肃农业大学生命科学技术学院
3. 甘肃农业大学动物科学技术学院
4. 河南大学生命科学学院
Generation of Asialoglycoprotein Receptor 1 (ASGR1) Gene Knockout Pigs (Sus scrofa) Via CRISPR/Cas9
全文: PDF (3916 KB)   HTML (1 KB) 
输出: BibTeX | EndNote (RIS)      
摘要 猪(Sus scrofa)内皮细胞的去唾液酸糖蛋白受体1(asialoglycoprotein receptor 1, ASGR1)是诱发异种肝移植后受体发生血小板减少症的主要诱因之一。本研究在α-1, 3-半乳糖基转移酶基因(α-1, 3-galactosyltransferase, GGTA1)敲除的五指山小型猪基础上,利用规律成簇间隔短回文重复(clustered regularly interspaced short palindromic repeats/Cas9, CRISPR/Cas9)结合体细胞核移植技术制备ASGR1基因敲除猪,针对猪ASGR1基因设计并构建了靶向表达载体单链导向RNA1 (single guide RNA1, sgRNA1),将sgRNA1与增强绿色荧光蛋白(enhanced green fluorescent protein, EGFP)质粒共同电转染GGTA1基因敲除猪耳成纤维细胞,流式细胞仪富集筛选带绿色荧光的细胞后培养单细胞克隆。实验结果表明,PCR测序检测培养获得的41个单细胞克隆,37个克隆发生基因突变,ASGR1基因突变效率为90%,其中双等位基因敲除的细胞克隆30个,效率高达73%。选择4个ASGR1基因敲除类型不同的细胞为核供体进行核移植,将早期重构胚移植到4头受体猪,2头妊娠至终期,产仔猪6头,Western blot显示ASGR1基因敲除仔猪的肝脏中没有ASGR1的表达。对sgRNA1的13个潜在脱靶位点,分别设计引物进行PCR扩增和测序,结果显示没有脱靶现象。总之,本研究利用CRISPR/Cas9结合体细胞核移植技术快速高效地制备了GGTA1/ASGR1基因敲除五指山小型猪模型,有望解决异种肝移植后出现的血小板减少症,为临床过渡肝的应用研究提供了良好的研究材料。
服务
把本文推荐给朋友
加入我的书架
加入引用管理器
E-mail Alert
RSS
作者相关文章
李西睿
冯冲
龙川
纪慧丽
魏静
石宁宁
曾国敏
蒋应弟
潘登科
田兴华
关键词 规律成簇间隔短回文重复(CRISPR/Cas9)去唾液酸糖蛋白受体1(ASGR1)异种肝移植体细胞核移植基因敲除猪    
Abstract:Lethal thrombocytopenia that accompanies liver xenotransplantation is one of the barrier to clinical application. Asialoglycoprotein receptor 1 (ASGR1) in pig (Sus scrofa) sinusoidal endothelial cells can bind and phagocytose human (Homo sapiens) platelets, causing thrombocytopenia. ASGR1 knockout pigs were generated by clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) in α-1,3-galactosyltransferase (GGTA1) gene knockout pigs, which could avoid hyperacute rejection and diminish platelet destruction in pig-to-human xenotransplantation. Single guide RNA1 (sgRNA1) was assembled and transferred to ?broblasts with enhanced green fluorescent protein (EGFP) plasmid. Forty-one colonies were finally selected, and the targeting efficiency reached 90% (37/41), more remarkably 73% (30/41) of colonies were biallelic knockout, among which 4 colonies with different genotypes were selected as nuclear donors for somatic cell nuclear transfer (SCNT). Cloned embryos were co-transferred into four recipient gilts. Pregnancy was established in 2 of 4 transfers. Two pregnancies were maintained to term, resulting in 6 live-born piglets with biallelic mutations of the ASGR1 gene. Western blot results showed that the ASGR1 gene expression was abrogate in the liver. Thirteen potential off-target sites were investigated, but no off-target event was detected in the live piglets. Taken together, the use of the CRISPR/Cas9 system of fibroblasts followed by SCNT enabled the production of ASGR1 biallelic knockout pigs. Thus, deletion of the ASGR1 gene is a viable strategy to diminish platelet destruction in pig-to-human xenotransplantation.
Key wordsClustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9)    Asialoglycoprotein receptor 1 (ASGR1)    Xenotransplantation    Somatic cell nuclear transfer (SCNT)    Knockout pig
收稿日期: 2016-03-16      出版日期: 2016-07-01
基金资助:国家重点基础研究发展计划
通讯作者: 田兴华     E-mail: tianxh@henu.edu.cn
引用本文:   
李西睿 冯冲 龙川 纪慧丽 魏静 石宁宁 曾国敏 蒋应弟 潘登科 田兴华. CRISPR/Cas9介导的ASGR1基因敲除猪制备[J]. , 2016, 24(8): 1243-1250.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I8/1243
 
版权所有 © 2014 《农业生物技术学报》编辑部   京ICP备11035905号-3
地址:北京市海淀区圆明园西路2号中国农业大学生命科学楼1053室 邮编:100193
电话:010-62733684 传真:010-62731615 E-mail: nsjxb@cau.edu.cn