关键词:

Abstract:

Objective To obtain α-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 forestablishing Bama miniature pigs with α-Gal and SD（a）antigen deficiency. MethodsSingle-guide RNA（sgRNA）targeting the third exon of pig GGTA1 and the eighth exon of pig β4GalNT2 weredesigned 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 wereco-transfected withaneomycin-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 and β4GalNT2 gene were successfully constructed and transfected into PFFs. Among the 48 G418-resistant colonies obtained，five hadbiallelic modifications in both GGTA1 and β4GalNT2 loci. Conclusion The GGTA1/β4GalNT2 double gene knockout PFFs were successfully obtained by the highly efficient CRISPR/Cas9 targeting，which could contribute tothe generation of GGTA1/β4GalNT2-deficient Bama miniature pig models.

Key words:

Bama miniature pigs ；CRISPR/Cas9 ；porcine fetal fibroblasts ；α-1, 3-galactosyltransferase/β-1,

4 Nacetylgalactosaminyltransferase