Analyzing bovine OCT4 and NANOG enhancer activity in pluripotent stem cells using fluorescent protein reporters

PLoS One. 2018 Oct 5;13(10):e0203923. doi: 10.1371/journal.pone.0203923. eCollection 2018.

Abstract

Green fluorescent protein (GFP) reporters controlled by the regulatory region of OCT4 and NANOG-two master regulators for pluripotency are widely used in studies of pluripotent stem cell establishment and embryo development. Alongside the challenge in establishing bovine pluripotent stem cells, the application of bovine-specific gene reporters has rarely been explored. Using lentivirus-based GFP reporter, we investigated the upstream regulatory regions of bovine OCT4 and NANOG. These reporters show activity in both naïve- and primed-state pluripotency when infected into mouse and human embryonic stem cells (ESCs), respectively. Consistent with what is found in humans and mice, the bovine OCT4-distal enhancer (bOCT4-DE) but not the proximal enhancer (bOCT4-PE) region is preferentially activated in naïve-state pluripotency. Furthermore, the bOCT4-DE region is silenced upon conversion of naive-state ESCs into primed-state epiblast stem cells (EpiSCs). Co-infection of mouse fibroblasts with the reprograming factors for induced pluripotent stem cell (iPSC) induction leads to the generation of GFP positive colonies, demonstrating that these GFP reporters can serve as live indicators for induced pluripotent cell establishment. We further proved that the bovine OCT4 distal enhancer is active in bovine blastocysts. We established the lentiviral-based fluorescent reporters controlled by bovine OCT4 and NANOG enhancer sequences. These reporter constructs show activity in naïve- and primed-pluripotent states. These reporters may serve as versatile tools for bovine ESC/iPSC generation and identification, as well as for developmental studies of bovine embryos.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cattle
  • Cells, Cultured
  • Enhancer Elements, Genetic*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • Human Embryonic Stem Cells / cytology
  • Human Embryonic Stem Cells / metabolism
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / metabolism
  • Lentivirus / genetics
  • Mice
  • Mouse Embryonic Stem Cells / cytology
  • Mouse Embryonic Stem Cells / metabolism
  • Nanog Homeobox Protein / genetics*
  • Octamer Transcription Factor-3 / genetics*
  • Pluripotent Stem Cells / cytology
  • Pluripotent Stem Cells / metabolism*
  • Transcriptional Activation

Substances

  • Nanog Homeobox Protein
  • Octamer Transcription Factor-3
  • Green Fluorescent Proteins

Grants and funding

This work was supported by the United States Department of Agriculture (USDA) W2171 regional project to Y.T., the Agriculture and Food Research Initiative Competitive Grant no. 2016-67016-24894 to Y.T. from the USDA National Institute of Food and Agriculture (https://nifa.usda.gov/), and the Chinese Scholarship Council for Study Abroad for D.H. and M.Z. (https://www.csc.edu.cn/chuguo).