利用CRISPR/Cas9系统构建猪PLIN1基因敲除载体及其效率检测  

Construction of Porcine PLIN 1 Gene Knockout Vector Using CRISPR/Cas9 System and its Efficiency Detection

作　　者：彭玥晗 徐磊[1] 许金蔓 王昊 胡悦旻 鞠辉明[1,2] PENG Yue-han;XU Lei;XU Jin-man;WANG Hao;HU Yue-min;JU Hui-ming(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Collaborative Innovation Center for Animal Epidemics and Zoonoses,Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医杂志》2021年第4期1-4,I0013,共5页Chinese Journal of Veterinary Medicine

基　　金：江苏高校优势学科建设工程资助项目(2020);国家自然科学基金项目(31872323);江苏高校品牌专业建设工程资助项目(TAAP,2018-2022)。

摘　　要：围脂滴蛋白(PLIN1)是具有抑制基础脂解,调节脂肪甘油三酯的储存和水解的蛋白。本试验利用CRISPR/Cas9系统设计了3条能够靶向切割PLIN 1基因的sgRNA并构建相应的突变载体。将构建好的质粒转染入PK15细胞,利用嘌呤霉素筛选,富集PLIN 1突变细胞,提取各组细胞DNA进行PCR扩增突变区域,通过序列测定在DNA水平确定突变效率。提取各组细胞总RNA及蛋白,分别利用qRT-PCR及Western Blot测定PLIN1 RNA水平和蛋白水平的表达量。结果表明,各组细胞PLIN 1 DNA均检测到突变或缺失,PLIN1-KO1、PLIN1-KO2和PLIN1-KO3突变比率分别为8%、30%和18%,且mRNA及蛋白表达量均有下降,其中PLIN1-KO2突变效率最高,RNA和蛋白表达量与正常组细胞相比分别下降78.26%和74.00%,差异均极显著(P<0.01)。本试验成功构建了PLIN 1基因敲除载体,其中PLIN1-KO2载体突变效率最高,为后续研究PLIN 1基因对猪肌肉发育、能量代谢及线粒体功能等方面的作用提供科学依据。Perilipin1(PLIN1)is a protein that inhibits basic lipolysis and regulates the storage and hydrolysis of fatty triglycerides.In this study,the CRISPR/Cas9 system was used to design 3 sgRNA that can target to cut PLIN 1 gene and construct corresponding mutation vectors.The constructed plasmid was transfected into PK15 cells,and PLIN 1 mutation cells were enriched by purinamycin screening.The DNA of each group was extracted for PCR amplification of mutation regions,and the mutation efficiency was determined at the DNA level by sequencing.Total RNA and protein of each group were extracted and the expression of PLIN 1 was determined by qRT-PCR and Western Blot,respectively.The results showed that mutation or deletion of PLIN 1 DNA was detected in all groups,and the mutation ratios of PLIN1-KO1、PLIN1-KO2、PLIN1-KO3 were 8%,30%and 18%,respectively.The mRNA and protein expressions of the three groups all decreased,among which PLIN1-KO2 mutation efficiency was the highest,and RNA and protein expressions decreased by 78.26%and 74.00%,respectively,compared with the normal group,the differences were all very significant(P<0.01).In this study,PLIN 1 gene knockout vector was successfully constructed,in which PLIN1-KO2 vector mutation efficiency was the highest,laying a foundation for subsequent studies on the effects of PLIN 1 gene on muscle development,energy metabolism and mitochondrial function in pigs.

关 键 词：猪 PLIN 1 CRISPR/Cas9 基因突变 

分 类 号：R730.2[医药卫生—肿瘤]