同源臂长度对CRISPR/Cas9介导hLF基因打靶山羊β-乳球蛋白位点效率的影响  

Effects of homologous arm length on the efficiency of CRISPR/Cas9 mediated hLF gene knock-in at goatβ-lactoglobulin locus

作　　者：李丹 周鸣鸣 何正义 吴赵曼秋 宋绍征 LI Dan;ZHOU Ming-ming;HE Zheng-yi;WU Zhao-man-qiu;SONG Shao-zheng(Department of Basic Medicine,School of Health and Nursing,Wuxi Taihu University,Wuxi,Jiangsu 214000,China;College of Veterinary Medicine,Yangzhou University/Jiangsu Provincial Research Center for Animal Transgenesis and Biopharming,Yangzhou,Jiangsu 225009,China)

机构地区：[1]无锡太湖学院健康与护理学院基础医学系,江苏无锡214000 [2]扬州大学兽医学院/江苏省转基因动物制药工程研究中心,江苏扬州225009

出　　处：《南方农业学报》2022年第1期182-190,共9页Journal of Southern Agriculture

基　　金：国家重点研发计划项目(2016YFE0126000);江西省卫生健康委员会科技计划项目(202130627);江苏省高等学校自然科学研究面上项目(19KJB180030);江苏省高校“青蓝工程”优秀青年骨干教师项目(苏教师函〔2021〕11号)。

摘　　要：【目的】探究同源臂长度对CRISPR/Cas9系统介导人乳铁蛋白基因(hLF)打靶山羊β-乳球蛋白基因(BLG)座位点效率的影响,为今后体细胞核移植制备BLG-/hLF+基因打靶山羊提供科学依据,也为CRISPR/Cas9基因编辑系统介导BLG基因或其他基因座位点定向精准分子修饰的遗传育种提供借鉴。【方法】针对山羊BLG基因第一外显子区域设计构建sgBLG/Cas9载体,电转染山羊胎儿成纤维细胞,PCR验证BLG基因座位点致突变活性;以BLC14乳腺特异性表达载体为基础构建3种同源臂长度(6.0、3.5和1.2 kb)的hLF基因打靶载体,分别与sgBLG/Cas9载体共转染山羊胎儿成纤维细胞,经500μg/mLG418筛选后,采用PCR检测基因打靶情况。【结果】sgBLG/Cas9载体在山羊胎儿成纤维细胞BLG基因座附近切割DNA双链的致突变活性效率在30%~35%。构建获得3种同源臂长度的hLF基因打靶载体(BLC14-1、BLC14-2和BLC14-3),对应的同源臂长度分别为6.0、3.5和1.2 kb;将3种hLF基因打靶载体与sgBLG/Cas9载体共转染山羊胎儿成纤维细胞,经5次电转染和G418筛选,分别获得83、77和86株药物抗性细胞,经PCR同源重组检测最终获得42、38和44株BLG-/hLF+基因打靶细胞株,即hLF基因在山羊胎儿成纤维细胞BLG基因座的平均打靶效率分别为50.6%(42/83)、49.4%(38/77)和51.2%(44/86)。3种不同长度同源臂构建的hLF基因打靶载体在山羊BLG基因座位点的打靶效率在统计学上无显著差异(P>0.05),表明同源臂长度对CRISPR/Cas9介导hLF基因打靶山羊BLG基因座位点无显著影响。【结论】利用CRISPR/Cas9系统介导hLF基因打靶山羊胎儿成纤维细胞BLG基因座位点能成功获得多株hLF+/BLG-基因打靶细胞株(BLG基因座定点打靶hLF基因),但打靶载体同源臂长度对CRISPR/Cas9系统介导BLG位点定向整合hLF基因的打靶效率无明显影响。【Objective】To explore the effect of homology arm length on the efficiency of CRISPR/Cas9 system-mediated human lactoferrin gene(hLF)targeting goatβ-lactoglobulin gene(BLG)locus,and to provide a scientific basis for the preparation of BLG-/hLF+gene target cells by somatic cell nuclear transfer in the future.It also provided a reference for genetic breeding of BLG gene or other gene locus directed by precise molecular modification mediated by CRISPR/Cas9 gene editing system.【Method】According to the first exon region of the goat BLG gene,the sgBLG/Cas9 vector was designed and constructed,and it was electrotransfected goat fetal fibroblasts.The site-mutagenic activity of the BLG gene locus was verified by PCR.Based on BLC14 mammary gland-specific expression vector,three hLF gene targeting vectors with homology arm length(6.0,3.5 and 1.2 kb)were constructed,and it was cotransfected into goat fetal fibroblasts with sgBLG/Cas9 vector respectively.After 500µg/mL G418 screening,the gene targeting was detected by PCR.【Result】The mutagenic activity efficiency of cleaving DNA double strands with BLG locus in goat fetal fibroblasts was 30%-35%by sgBLG/Cas9 vector.The hLF gene targeting vectors(BLC14-1,BLC14-2 and BLC14-3)were constructed to obtain three homology arm lengths,corresponding to 6.0,3.5 and 1.2 kb.Three hLF gene targeting vectors and sgBLG/Cas9 vector were cotransfected into goat fetal fibroblasts.After 5 times of electrotransfections and G418 screening,83,77 and 86 drug-resistant cells were obtained respectively.Finally,42,38 and 44 BLG-/hLF+gene targeting cell lines were obtained by PCR homologous recombination detection.The average targeting efficiencies of hLF gene at the BLG locus of goat fetal fibroblasts were 50.6%(42/83),49.4%(38/77)and 51.2%(44/86).There was no statistically significant difference in the targeting efficiency of hLF gene targeting vectors constructed with three different lengths of homology arms at the BLG gene locus in goats(P>0.05),indicating that the homology arm length ha

关 键 词：山羊 CRISPR/Cas9 基因打靶 同源臂长度 β-乳球蛋白基因(BLG) 人乳铁蛋白基因(hLF) 

分 类 号：S814.8[农业科学—畜牧学]