Efficient bi-allelic gene knockout and site-specific knock-in mediated by TALENs in pigs

Objective To establish the platform of TALENs mediated gene editing in pigs and produce DJ-1 gene knockout(KO) pigs.Methods TALENs against GGTA1,Parkin and DJ-1 genes were co-transfected respectively into porcine fibroblast cells with pCAG-GFP plasmid to obtain gene KO colonies.And single-strand oligodeoxynucleotides(ssODN) ontaining Hindlll restriction enzyme site was co-transfected with TALENs against DJ-1 gene to test the possibility of TALEN-mediated site-specific knockin.Single colonies were screened by FACS,and targeted cell colonies were identified by PCR,enzyme digestion and sequencing.Further,mixed DJ-1 mutant colonies were used as donor cells for somatic cell nuclear transfer(SCNT) to produce DJ-1 KO pigs.Results In total,5%(2/40),2.5%(2/80),and 22%(11/50) of the obtained colonies of fibroblast cells were mutated for GGTA1,Parkin,and DJ-1,respectively.Among these mutant colonies,over 1/3were bi-allelic KO,and no off-target cleavage was detected.One of the ten mutant colonies had the Hindlll site ins...Objective To establish the platform of TALENs mediated gene editing in pigs and produce DJ-1 gene knockout(KO) pigs.Methods TALENs against GGTA1,Parkin and DJ-1 genes were co-transfected respectively into porcine fibroblast cells with pCAG-GFP plasmid to obtain gene KO colonies.And single-strand oligodeoxynucleotides(ssODN) ontaining Hindlll restriction enzyme site was co-transfected with TALENs against DJ-1 gene to test the possibility of TALEN-mediated site-specific knockin.Single colonies were screened中国实验动物学会