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

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，2 μg/mL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700 μg/mL G418 和2 μg/mL 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。

Abstract:

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/mL 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/mL G418 and 2 μg/mL 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.