CRISPR/Cas9系统介导的人乳铁蛋白基因在山羊β-乳球蛋白基因座定点敲入  

hLF gene knock-in at the BLGlocus of goat by CRISPR/Cas9 system

作　　者：宋绍征 张婷[2] 潘生强 陆睿 成勇[2] 周鸣鸣 SONG Shaozheng;ZHANG Ting;PAN Shengqiang;LU Rui;CHENG Yong;ZHOU Mingming(School of Nursing,Wuxi Taihu University,Wuxi 214000,China;College of Veterinary Medicine/Jiangsu Provincial Research Center for Animal Transgenesis and Biopharming,Yangzhou University,Yangzhou 225009,China;School of Pharmaceutical Engineering,Jiangsu Food and Pharmaceutical Science College,Huaian 223003,China)

机构地区：[1]无锡太湖学院护理学院,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 [3]江苏食品药品职业技术学院制药工程学院,江苏淮安223003

出　　处：《中国农业大学学报》2020年第7期111-119,共9页Journal of China Agricultural University

基　　金：国家转基因生物新品种培育重大专项(2014ZX08008-004);江苏省高校自然科学基金面上项目(19KJB180030);无锡市科协软科学研究重点课题(KX-19-B28)。

摘　　要：为研究山羊乳蛋白基因编辑,实现羊乳"人源化"改造。利用CRISPR/Cas9系统敲除山羊乳中致敏原β-乳球蛋白(BLG)基因,同时在BLG基因座定点敲入人乳铁蛋白(hLF)基因。针对山羊BLG基因第一外显子设计构建sgBLG/Cas9表达载体经电转染山羊胎儿成纤维细胞验证其编辑活性;将打靶载体BLC14与sgBLG/Cas9载体共转染山羊胎儿成纤维细胞,经筛选检测获得BLG^-/hLF^+打靶细胞株作为供核细胞用于山羊体细胞核移植;通过剖宫产获取克隆胎儿并验证其中靶情况。结果表明:sgBLG/Cas9编辑山羊BLG位点致突变率约为30%~35%;共筛选72株药物抗性细胞株,其中有37株为基因打靶细胞,打靶效率为51.4%(37/72);挑取形态较好的7株打靶细胞用于核移植,共制备416枚重构胚,移植30只受体山羊;30-35日龄B超诊断有13只受孕,妊娠率为43.3%(13/30);剖宫产获取3只35日龄克隆胎儿均验证为BLG^-/hLF^+基因型,并建立了BLG^-/hLF^+靶向性修饰细胞系。因此,利用CRISPR/Cas9系统可以获得BLG基因座定点打靶hLF基因的转基因克隆山羊,该研究为培育羊乳中低致敏原和富含hLF功能营养成分的转基因山羊新品系提供了科学依据。In order to study the gene editing of goat milk protein and realize the"humanization"transformation of goat milk,theβ-lactoglobulin(BLG)gene was knocked out and the human lactoferrin(hLF)gene was knocked in at BLG locus in goat by CRISPR/cas9 system.A sgBLG/Cas9 expression vector for goat BLG exonⅠrecognition site was designed and constructed,which was electrotransfected into the goat fetal fibroblast cells to verify the editing activity.The vector BLC14 and sgBLG/Cas9 were co-transfected into goat fetal fibroblasts,and the BLG^-/hLF^+targeting cells were chosen as donor cells for goat somatic cell nuclear transfer.The cloned fetus was obtained by cesarean section and the target condition was verified.The results showed that:The mutagenicity of sgBLG/Cas9 editing goat BLG locus was 25%-30%;A totoal of 72 G418-resistant cells were screened,37 of which were gene targeting cells,and the targeting efficiency was 51.4%(37/72);In this study,seven gene target cells having better morphology appearances were selected for nuclear transplantation,and 416 reconstructed embryos were transferred into 30 recipient goats.There were 13 pregnancies confirmed by B-ultrasound in 30-35 days,and pregnancy rate was 43.3%(13/30).All three 35-day-old cloned fetuses by cesarean section,and they were all identified as BLG^-/hLF^+genotype and the targeted modified cell lines were established.In conclusion,the hLF gene knock-in at BLGlocus of goat could be obtained by CRISPR/cas9 system,which provided a scientific basis for cultivating transgenic goat lines of low allergen and rich hLF in the milk,and also laid a foundation for milk protein gene editing and molecular genetic breeding.

关 键 词：CRISPR/Cas9 Β-乳球蛋白 人乳铁蛋白 基因打靶 核移植 

分 类 号：S857.21[农业科学—临床兽医学]