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

Stem Cell Research

Bálint László Bálint¹  Renáta Bencsik²  Pal Boto²  Renáta Nóra Szabó²  Bianka Monika Toth²  Istvan Szatmari²  Emilia Simo² 

[1] Genomic Medicine and Bioinformatic Core Facility, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen H-4010, Hungary;Stem Cell Differentiation Laboratory, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen H-4010, Hungary;

关键词: Inducible cells;    Reporter genes;    mCherry;    Mouse embryonic stem cells;    Transgenic cells;   

DOI  :  10.1016/j.scr.2016.08.014

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown