转录激活因子样效应物核酸酶介导的山羊β-乳球蛋白基因敲除和人乳铁蛋白基因定点整合  

BLG gene knockout and hLF gene knock-in at BLG locus in goat by TALENs

作　　者：宋绍征[1,2] 朱孟敏 袁玉国[1,2] 荣耀[1] 徐晟[1] 陈思[1] 梅珺琰 成勇[1,2] 

机构地区：[1]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 [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：《生物工程学报》2016年第3期329-338,共10页Chinese Journal of Biotechnology

基　　金：国家转基因生物新品种培育重大专项(No.2011ZX08008-004);江苏高校优势学科建设工程资助项目;扬州大学研究生科研创新计划项目(No.CXLX-1435)资助~~

摘　　要：为了敲除山羊乳中致敏源β-乳球蛋白(BLG)基因,同时在BLG基因座定点整合人乳铁蛋白(hLF)基因。首先针对山羊BLG第3外显子识别位点设计了1对特异性TALEN-3-L/R质粒对;同时,构建了含有1个HSV-TK负筛选基因的hLF基因打靶载体BLC14-TK。TALENs质粒对转染山羊胎儿成纤维细胞,2μg/m L嘌呤霉素筛选3 d,PCR扩增产物测序来验证其切割DNA活性。打靶载体BLC14-TK与TALEN-3-L/R质粒对共转染山羊胎儿成纤维细胞,经700μg/m L G418和2μg/m L GCV共筛选药物抗性细胞株;通过整合检测和同源重组检测来筛选hLF基因打靶细胞株;BLG~–/hLF^+打靶细胞株作为供核细胞进行山羊体细胞核移植。结果为:TALEN-3-L/R致突变率为25%-30%;获得BLG~–/hLF^+打靶细胞6株;共制作重构胚胎335枚,移植受体山羊23只,B超检测到30-35 d的妊娠受体9只(妊娠率39.1%),其中1只50日龄克隆胎儿验证为BLG~–/hLF^+基因型。以上结果表明获得BLG基因座定点整合hLF基因的基因打靶山羊是可行的,为培育羊乳中含低致敏原和富含hLF的山羊新品系奠定了基础。To knock out β-lactoglobulin（BLG） gene and insert human lactoferrin（hLF） coding sequence at BLG locus of goat, the transcription activator-like effector nucleases（TALEN） mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon Ⅲ recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/m L puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/m L G418 and 2 μg/m L GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG~–/hLF~＋ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35（pregnancy rate of 39.1%）, and one of 50-day-old fetus with BLG~–/hLF~＋ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.

关 键 词：TALENs 人乳铁蛋白 BLG 基因打靶 体细胞核移植 

分 类 号：Q78[生物学—分子生物学] S827[农业科学—畜牧学]