Oncotarget

Research Papers:

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

Jin-Dan Kang, Hyojin Kim, Long Jin, Qing Guo, Cheng-Du Cui, Wen-Xue Li, Seokjoong Kim, Jin-Soo Kim _ and Xi-Jun Yin

PDF  |  HTML  |  How to cite  |  Order a Reprint

Oncotarget. 2017; 8:115480-115489. https://doi.org/10.18632/oncotarget.23301

Metrics: PDF 381 views  |   HTML 1087 views  |   ?  


Abstract

Jin-Dan Kang1,*, Hyojin Kim2,4,*, Long Jin1,*, Qing Guo1, Cheng-Du Cui1, Wen-Xue Li1, Seokjoong Kim3, Jin-Soo Kim2 and Xi-Jun Yin1

1Jilin Provincial Key Laboratory of Transgenic Animal and Embryo Engineering and Department of Animal Science, Yanbian University, Yanji 133002, China

2Center for Genome Engineering, Institute for Basic Science, Gwanak-gu, Seoul 151-747, South Korea

3ToolGen, Inc., Byucksan Kyoungin Digital Valley 2-Cha, Geumcheon-Gu, Seoul 153-023, South Korea

4Present/Current address: Department of Biosystems Science and Engineering, ETH Zurich, Basel CH-4058, Switzerland

*These authors have contributed equally to this work

Correspondence to:

Jin-Soo Kim, email: jskim01@snu.ac.kr

Xi-Jun Yin, email: yinxj33@msn.com

Keywords: PDX1; TALEN; SCNT; pancreas; pig

Received: September 30, 2017     Accepted: December 05, 2017     Published: December 14, 2017

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.


Creative Commons License All site content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 License.
PII: 23301