Improved transgene expression in doxycycline-inducible embryonic stem cells by repeated chemical selection or cell sorting

Renáta Bencsik, Pal Boto, Renáta Nóra Szabó, Bianka Monika Toth, Emilia Simo, Bálint László Bálint, Istvan Szatmari

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Transgene-mediated programming is a preeminent strategy to direct cellular identity. To facilitate cell fate switching, lineage regulating genes must be efficiently and uniformly induced. However, gene expression is often heterogeneous in transgenic systems. Consistent with this notion, a non-uniform reporter gene expression was detected in our doxycycline (DOX)-regulated, murine embryonic stem (ES) cell clones. Interestingly, a significant fraction of cells within each clone failed to produce any reporter signals upon DOX treatment. We found that the majority of these non-responsive cells neither carry reporter transgene nor geneticin/G418 resistance. This observation suggested that our ES cell clones contained non-recombined cells that survived the G418 selection which was carried out during the establishment of these clones. We successfully eliminated most of these corrupted cells with repeated chemical (G418) selection, however, even after prolonged G418 treatments, a few cells remained non-responsive due to epigenetic silencing. We found that cell sorting has been the most efficient approach to select those cells which can uniformly and stably induce the integrated transgene in this ES cell based platform. Together, our data revealed that post-cloning chemical re-selection or cell sorting strongly facilitate the production of ES cell lines with a uniform transgene induction capacity.

Original languageEnglish
Pages (from-to)228-234
Number of pages7
JournalStem Cell Research
Volume17
Issue number2
DOIs
Publication statusPublished - Sep 1 2016

    Fingerprint

Keywords

  • Inducible cells
  • Mouse embryonic stem cells
  • Reporter genes
  • Transgenic cells
  • mCherry

ASJC Scopus subject areas

  • Developmental Biology
  • Cell Biology

Cite this