利用CRISPR/Cas9技术制备BLG基因敲除牛乳腺上皮细胞系  

Highly Efficient BLG Knockout in Bovine Mammary Epithelial Cells by Using CRISPR/Cas9

作　　者：丁修虎 林志平 赵芳[1] 陈坤琳 仲跻峰 张燕 高运东 李惠侠[2] 王慧利[1] 张建丽[1] 丁强 DING Xiuhu;LIN Zhiping;ZHAO Fang;CHEN Kunlin;ZHONG Jifeng;ZHANG Yan;GAO Yundong;LI Huixia;WANG Huili;ZHANG Jianli;DING Qiang(Key Laboratory of Crop and Animal Integrated Farming of Ministry of Agriculture and Rural Affairs,Jiangsu Province Engineering Research Center for Precision Animal Breeding,Institute of Animal Science,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;College of Animal Science&Technology,Nanjing Agricultural University,Nanjing 210095,China;Jiangsu Youyuan Dairy Research Institute Co.,Ltd.,Nanjing 211100,China;Shandong OX Livestock Breeding Co.,Ltd.,Jinan 250100,China)

机构地区：[1]江苏省农业科学院畜牧研究所,江苏省畜禽精准育种工程研究中心,农业农村部种养结合重点实验室,南京210014 [2]南京农业大学动物科技学院,南京210095 [3]江苏优源奶业产业研究院有限公司,南京211100 [4]山东奥克斯畜牧种业有限公司,济南250100

出　　处：《畜牧兽医学报》2024年第10期4475-4488,共14页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：江苏省重点研发项目“基于新型单碱基编辑器的奶牛高效精准育种技术研究”(BE2021372);江苏省农业科学院探索性颠覆性创新计划项目(ZX(21)1214)。

摘　　要：旨在利用CRISPR/Cas9系统构建BLG基因敲除牛乳腺上皮细胞系(bovine mammary epithelial cells,bMECs),同时检测BLG基因敲除后对细胞的影响。本研究利用体外切割试验筛选获得靶向牛的BLG基因的第一外显子序列的3条sgRNA,与CRISPR/Cas9表达载体电转染入bMECs,利用浓度为1.8μg·mL^(-1)嘌呤霉素和6μg·mL^(-1)稻瘟菌素双药筛和PCR分析鉴定获得BLG基因编辑的单克隆细胞株;利用Western blot(WB)验证细胞BLG表达;绘制生长曲线和CCK-8试验检测BLG基因敲除对bMECs细胞增殖的影响;同时根据sgRNA序列,对基因组上潜在脱靶位点进行PCR测序分析。经筛选和PCR序列分析,共筛选获得了9株BLG基因编辑细胞株,其编辑效率为90%(9/10),其中4株细胞株BLG基因大片段删除。序列分析结果显示,单克隆细胞株编辑位点呈现片段插入缺失和碱基替换等多种编辑类型,WB结果显示敲除细胞株BLG蛋白表达显著降低,且细胞生长曲线和CCK-8检测结果表明BLG基因敲除的细胞生长速度加快。本研究利用CRISPR/Cas9技术成果构建了牛乳腺上皮细胞BLG基因高效敲除方法,BLG基因敲除后可显著影响牛乳腺上皮细胞的增殖,为探究BLG敲除牛在乳腺上皮细胞水平探究其功能机制提供细胞模型。This study aimed to establish CRISPR/Cas9 mediated BLG-knockout(BLG-KO)system in bovine mammary epithelial cells(bMECs),and to explore the function effect of BLG-KO on bMECs.Three sgRNAs were designed from the first exon of the bovine BLG gene sequence and screened by using in vitro cleavage test.The Cas9 expression vector and sgRNAs vectors were cotransfected into bMECs by electroporation.Monoclonal cell lines were screened with 1.8μg·mL^(-1)puromycin and 6μg·mL^(-1)blasticidin.BLG-KO gene editing type were verified by using PCR amplification and sequencing analysis.The potential off-target sites were selected according to the sgRNA sequence and detected by PCR sequencing.The BLG expressing level of knockout cell was detected by Western blot(WB).Cell viability was tested by cell counting kit-8(CCK-8)assay,further the growth curve was drawn to analyze the proliferation effect of bMECs cells after BLG-KO.Two sgRNAs were screened and used for further gene edit.10 monoclonal cell lines were obtained after screening and 9 BLG-edit lines verified by PCR sequencing,4 clones with large fragment deleted at BLG sequence.Sequence analysis showed that the editing sites contained multiple gene-editing types,including insertion,deletion and base substitution.WB result showed that BLG-KO cell lines with low BLG protein expressing level compare to none edit cell.Furthermore,the BLG-KO promote cell growth and proliferation according to the results of cell growth curve and CCK-8 assay.In this study,we constructed an efficient BLG-KO method in bMECs by using CRISPR/Cas9 system,and the results demonstrated that BLG-KO significantly affected the cell proliferation in bMECs,the results provided a cellular model for exploring the functional mechanism for BLG-KO cattle.

关 键 词：BLG基因 牛乳腺上皮细胞系 CRISPR/Cas9 基因编辑 

分 类 号：S823.2[农业科学—畜牧学]