Apancreatic pigs cloned using Pdx1-disrupted fibroblasts created via TALEN-mediated mutagenesis

Abstract

Pancreatic and duodenal homeobox 1 (PDX1) plays a crucial role in pancreas development, β-cell differentiation, and maintenance of mature β-cell function. In this study, we designed a strategy to produce PDX1-knockout (KO) pigs. A transcription activator-like effector nuclease (TALEN) pair targeting exon 1 of the swine PDX1 gene was constructed. Porcine fetal fibroblasts (PFFs) were transfected with the TALEN plasmids plus a surrogate reporter plasmid. PDX1-mutated PFFs were enriched by magnetic separation and used to produce homozygous PDX1-KO pigs via a two-step somatic cell nuclear transfer (SCNT) cloning process. In the first SCNT step, we obtained eight fetuses, established PFF cell lines, and analyzed PDX1 gene mutations by T7 endonuclease 1 assays and Sanger sequencing. Five fetuses showed mutations at the PDX1 loci with two biallelic mutations and three monoallelic mutations (mutation rate of 62.5%). In the second step, a PDX1 biallelic mutant PFF cell line with a 2 bp deletion in one allele and a 4 bp insertion in the other allele was used as a donor to generate cloned pigs via SCNT. From 462 cloned embryos transferred into two surrogates, nine live piglets were delivered. These piglets at birth were not clearly distinguishable phenotypically from wild-type piglets, but soon developed severe diarrhea and vomiting and all died within 2 days after birth. Dissection of PDX1-KO piglets revealed that the liver, gallbladder, spleen, stomach, common bile duct, and other viscera were present and normal, but the pancreas was absent in all cases.