Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering

PLoS One. 2016 Jan 12;11(1):e0146562. doi: 10.1371/journal.pone.0146562. eCollection 2016.

Abstract

The porcine pluripotent cells that can generate germline chimeras have not been developed. The Oct4 promoter-based fluorescent reporter system, which can be used to monitor pluripotency, is an important tool to generate authentic porcine pluripotent cells. In this study, we established a porcine Oct4 reporter system, wherein the endogenous Oct4 promoter directly controls red fluorescent protein (RFP). 2A-tdTomato sequence was inserted to replace the stop codon of the porcine Oct4 gene by homogenous recombination (HR). Thus, the fluorescence can accurately show the activation of endogenous Oct4. Porcine fetal fibroblast (PFF) lines with knock-in (KI) of the tdTomato gene in the downstream of endogenous Oct4 promoter were achieved using the CRISPR/CAS9 system. Transgenic PFFs were used as donor cells for somatic cell nuclear transfer (SCNT). Strong RFP expression was detected in the blastocysts and genital ridges of SCNT fetuses but not in other tissues. Two viable transgenic piglets were also produced by SCNT. Reprogramming of fibroblasts from the fetuses and piglets by another round of SCNT resulted in tdTomato reactivation in reconstructed blastocysts. Result indicated that a KI porcine reporter system to monitor the pluripotent status of cells was successfully developed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Newborn
  • Blastocyst / metabolism
  • CRISPR-Cas Systems / genetics*
  • Cellular Reprogramming
  • Fetus / cytology
  • Fibroblasts / metabolism
  • Gene Knock-In Techniques*
  • Genes, Reporter*
  • Genetic Engineering*
  • Genetic Vectors / metabolism
  • Genome*
  • Genotype
  • Octamer Transcription Factor-3 / genetics*
  • Promoter Regions, Genetic
  • Reproducibility of Results
  • Sus scrofa

Substances

  • Octamer Transcription Factor-3

Grants and funding

This work was supported in part by Strategic Priority Research Program of the Chinese Academy of Sciences (http://www.cas.cn/) Grant XDA010201070 and by Ministry of Science and Technology National Basic Research Program of China (http://www.most.gov.cn/index.htm) Grants 2011CB944204 and 2011CB944203. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.