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.

Generation of viable PDX1 gene‐edited founder pigs as providers of nonmosaics

“…Previous research has revealed that disruption of the PDX1 gene in pigs has lethal effects and PDX1 biallelic mutants showed severe health problems caused by the pancreatic agenesis, including growth retardation, dehydration, severe diarrhea, and vomiting (Kang et al, 2017). In this study, we observed that piglets carrying PDX1 biallelic mutation similarly showed severe health problems and died within 2 days of birth.…”

“…In the previous study, PDX1 ‐modified pigs have been generated by SCNT, which strategy is commonly used to generate mutant pigs (Kang et al, 2017). Although SCNT enables to generate mutant pigs carrying desired mutations, the use of this technique is associated with small litter sizes, low viability, and abnormal epigenetic regulation of resulting offsprings (Kurome et al, 2013; Wan et al, 2016; Zhao, Whyte, & Prather, 2010).…”

“…Pancreatic duodenal homeobox 1 ( PDX1 , also known as IPF‐1 , IDX‐1 , and STF‐1 ) is a crucial gene for pancreas development during the fetal period (Bonal & Herrera, 2008; Jonsson, Carlsson, Edlund, & Edlund, 1994; McKinnon & Docherty, 2001). The biallelic mutation of PDX1 leads to agenesis of the pancreas both in mice (Jonsson et al, 1994) and in pigs (Kang et al, 2017; Wu, Vilarino et al, 2017), and results in infant death, whereas in mice, monoallelic disruption impairs insulin secretion from pancreatic β‐cells (Brissova et al, 2002). To our knowledge, however, viable PDX1 monoallelic mutants have not been investigated in pigs because PDX1 disrupted pigs were generated by somatic cell nuclear transfer (SCNT) with PDX1 biallelic mutant cells in the previous study (Kang et al, 2017).…”

“…The biallelic mutation of PDX1 leads to agenesis of the pancreas both in mice (Jonsson et al, 1994) and in pigs (Kang et al, 2017; Wu, Vilarino et al, 2017), and results in infant death, whereas in mice, monoallelic disruption impairs insulin secretion from pancreatic β‐cells (Brissova et al, 2002). To our knowledge, however, viable PDX1 monoallelic mutants have not been investigated in pigs because PDX1 disrupted pigs were generated by somatic cell nuclear transfer (SCNT) with PDX1 biallelic mutant cells in the previous study (Kang et al, 2017). In humans, a monoallelic 1 bp deletion in PDX1 (Pro63fsdelC) are known to be a nonsense mutation causing diabetes, including maturity‐onset diabetes of the young Type 4, which develops before age 25 with fasting hyperglycemia without insulin (Clocquet et al, 2000; Fajans et al, 2010; Stoffers, Ferrer, Clarke, & Habener, 1997; Stoffers, Stanojevic, & Habener, 1998).…”

Compensation of Disabled Organogeneses in Genetically Modified Pig Fetuses by Blastocyst Complementation

“…In our previous study (Matsunari et al, 2013), we demonstrated that an apancreatic phenotype in pigs could be achieved by overexpressing the mouse Pdx1-Hes1 transgene. In this study, we knocked out porcine endogenous PDX1 gene, based on previous reports demonstrating apancreatic phenotype caused by Pdx1 À/À mutation in rodents (Jonsson et al, 1994;Kobayashi et al, 2010;Offield et al, 1996) and pigs (Kang et al, 2017).…”