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 D J-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 HindⅢ 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 D J-1 mutant colonies were used as donor cells for somatic cell nuclear transfer (SCNT) to produce D J-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 D J-1,respectively.Among these mutant colonies,over 1/3 were bi-allelic KO,and no off-target cleavage was detected.One of the ten mutant colonies had the HindⅢ site insertion in the ssODN transfected cells.Three female piglets were obtained (two were bi-allelically mutated,and one was mono-allelically mutated) and Western blot analysis showed that the expression of the D J-1 protein was disrupted in KO piglets.Conclusions TALENs mediated gene targeting and site-specific knockin in pigs were established,and a combination of TALENs technology with SCNT can efficiently generate bi-allelic KO pigs without the integration of exogenous DNA.