GGTA1/β4GalNT2双基因敲除的巴马小型猪PFFs细胞系的建立

Establishment of Bama miniature pig PFFS with GGTA1/β4GalNT2 deficiency by double gene knockout

目的利用CRISPR/Cas9基因编辑技术建立巴马小型猪胚胎成纤维细胞(porcine fetal fibroblasts,PFFs)α-1,3-半乳糖基转移酶(α-1,3-galactosyltransferase,GGTA1)/β-1,4 N-乙酸氨基半乳糖转移酶(β-1,4 N-acetylgalactosaminyltransferase,β4GalNT2)双基因敲除细胞系,为构建α-1,3-半乳糖(α-1,3-galactose,α-Gal)和SD(a)抗原缺失的巴马小型猪模型奠定基础。方法分别选取猪GGTA1基因的第三外显子和β4GalNT2基因的第八外显子为敲除靶点,利用在线工具(http://crispr.mit.edu)设计并合成单导向RNA(single guide RNA,sgRNA),以含有Cas9基因的pX330质粒为骨架构建打靶载体,转染野生巴马小型猪的PFFs,用T7EN1酶切验证打靶载体敲除效率。将打靶载体与G418抗性质粒(tdTomato)共转染巴马小型猪PFFs,经药物筛选获得阳性单细胞克隆后测序鉴定基因型。结果成功构建靶向GGTA1和β4GalNT2基因的Cas9/sgRNA表达载体。转染PFFs后用药物筛选得到31个单克隆细胞系,其中5个为GGTA1/β4GalNT2双基因敲除的细胞系。结论Cas9/sgRNA表达载体可以高效编辑PFFs的GGTA1/β4GalNT2基因并获得了双基因敲除的单细胞克隆,为构建GGTA1/β4GalNT2敲除的巴马小型猪提供必需的实验材料。

Objective To obtainα-1,3-galactosyltransferase/β-1,4 N-acetylgalactosaminyltransferase(GGTA1/β4GalNT2)double gene knockout porcine fetal fibroblasts(PFFs)by CRISPR/Cas9 system and lay the foundation for establishing Bama miniature pigs withα-Gal and SD(a)antigen deficiency.Methods Single-guide RNA(sgRNA)targeting the third exon of pig GGTA1 and the eighth exon of pigβ4GalNT2 were designed using online tools(http://crispr.mit.edu),respectively.The synthesized sgRNA were cloned into pX330 plasmid containing Cas9 skeleton.The cleavage efficiency of the Cas9/sgRNA plasmids was assessed by the T7EN1 enzyme digestion assay.The Cas9/sgRNA vectors were co-transfected with a neomycin-expression plasmid(tdTomato)into the PFFs.G418 was used to screen the positive monoclonal cells and sanger sequencing was used to determine the genotypes of monoclonal cells.Results Cas9/sgRNA expression vectors targeting GGTA1 andβ4GalNT2 gene were successfully constructed and transfected into PFFs.Among the 48 G418-resistant colonies obtained,five had biallelic modifications in both GGTA1 and β4GalNT2 loci. Conclusion The GGTA1/β4GalNT2 double geneknockout PFFs were successfully obtained by the highly efficient CRISPR/Cas9 targeting, which could contribute tothe generation of GGTA1/β4GalNT2-deficient Bama miniature pig models.