利用CRISPR/Cas9系统构建猪PFKM基因定点突变细胞株  

Generation of cell strains containing point mutations in PFKM by CRISPR/Cas9 system

作　　者：许金蔓 徐磊[1] 彭玥晗 杨跃飞[1] 鞠辉明[1] XU Jinman;XU Lei;PENG Yuehan;YANG Yuefei;JU Huiming(Jiatigsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses/College of Veterinary Medicine Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院/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：《扬州大学学报（农业与生命科学版）》2020年第4期36-39,共4页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家自然科学基金资助项目(31872323);江苏高校优势学科建设工程项目(PAPD);江苏高校品牌专业建设工程项目(TAAP,2018-20121)。

摘　　要：肌型磷酸果糖激酶(PFKM)是作用于果糖-6-磷酸生成果糖-1,6-双磷酸的关键调节酶,在机体葡萄糖代谢、肌肉发育等多个生物过程中起关键作用。根据猪PFKM序列结构特点设计sgRNA序列并构建CRISPR/Cas9基因敲除质粒。敲除质粒转染猪PK15细胞并经嘌呤霉素筛选,极限稀释法筛选出单细胞克隆,提取各个细胞克隆DNA,利用PCR扩增敲除区域序列,通过序列测定确定是否敲除成功,利用实时荧光定量PCR及Western blot分别检测PFKM mRNA及蛋白质表达水平。结果表明:转染CRISPR/Cas9基因敲除质粒筛选到的单细胞克隆中,有2株细胞PFKM基因序列发生基因突变,其中1个克隆为碱基插入,另1个为碱基插入、替换及缺失;qRT-PCR定量检测结果表明,筛选到1株细胞PFKM mRNA表达量下降89.41%,差异极显著。Western blot检测结果表明,与正常细胞组相比,2组敲除细胞株中PFKM蛋白表达量分别下降78.77%、81.63%,差异显著。这一研究显示利用CRISPR/Cas9系统成功获得了2株PFKM基因敲除细胞株,为后续研究PFKM基因在猪肌肉发育、能量代谢及线粒体功能等方面的作用奠定了基础。Muscle-type phosphofructokinase(PFKM) is a key regulatory enzyme that converts fructose-6-phosphate to fructose-1,6-diphosphate and plays an important role in diverse biological processes such as glucose metabolism and muscle development. In this study, the sgRNA sequence was designed based on the PFKM sequence and the CRISPR/Cas9 plasmid vector of gene knockout was constructed. The plasmid vector of gene knockout was transfected to PK15 cell lines and single cell clones were screened with puromycin and limited dilution method. DNA of every cell clone was extracted and polymerase chain reaction(PCR) was used to amplify the sequence of knockout region. Sequencing was used to determine if they were removed. The PFKM mRNA and protein were detected with qRT-PCR and Western blot respectively. The results showed that, in the single cell clones obtained by transfection of CRISPR/Cas9 gene knockout plasmid vector, the PFKM gene sequences of two strains had gene mutation, including one clone with base insertion and the other one with base insertion, substitution and deletion. Moreover, the results of qRT-PCR indicated that the PFKM mRNA expression of one cell clone decreased by 89.41%, which was statistically significant(P<0.01). The results of Western blot suggested that, compared with normal cell group, PFKM protein expression of two knockout cell clones decreased by 78.77% and 81.63%, respectively, which was statistically significant. We obtained two PFKM gene knockout cell clones by CRISPR/Cas9 plasmid vector in this study. This study laid the foundation for the subsequent study of the function of PFKM gene in muscle development, energy metabolism and mitochondrial function in pigs.

关 键 词：猪 PFKM基因 CRISPR/Cas9 基因定点突变 

分 类 号：Q78[生物学—分子生物学]