Generation of induced pluripotent stem cells (iPSCs) stably expressing CRISPR-based synergistic activation mediator (SAM)

Stem Cell Res. 2016 Nov;17(3):665-669. doi: 10.1016/j.scr.2016.10.011. Epub 2016 Nov 17.

Abstract

Human fibroblasts were engineered to express the CRISPR-based synergistic activation mediator (SAM) complex: dCas9-VP64 and MS2-P65-HSF1. Two induced pluripotent stem cells (iPSCs) clones expressing SAM were established by transducing these fibroblasts with lentivirus expressing OCT4, SOX2, KLF4 and C-MYC. We have validated that the reprogramming cassette is silenced in the SAM iPSC clones. Expression of pluripotency genes (OCT4, SOX2, LIN28A, NANOG, GDF3, SSEA4, and TRA-1-60), differentiation potential to all three germ layers, and normal karyotypes are validated. These SAM-iPSCs provide a novel, useful tool to investigate genetic regulation of stem cell proliferation and differentiation through CRISPR-mediated activation of endogenous genes.

MeSH terms

  • Cell Differentiation
  • Cell Line
  • Cellular Reprogramming
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Embryoid Bodies / cytology
  • Embryoid Bodies / metabolism
  • Female
  • Fibroblasts / cytology
  • Genetic Vectors / genetics
  • Genetic Vectors / metabolism
  • HEK293 Cells
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / metabolism
  • Karyotype
  • Kruppel-Like Factor 4
  • Lentivirus / genetics
  • Microscopy, Fluorescence
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • KLF4 protein, human
  • Kruppel-Like Factor 4
  • Transcription Factors