title: Improved transgene expression in doxycycline-inducible embryonic stem cells by repeated chemical selection or cell sorting.
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.
authors: Stem Cell Differentiation Laboratory, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen H-4010, Hungary.
authors: Renáta
authors: R
authors: Bencsik
authors: Stem Cell Differentiation Laboratory, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen H-4010, Hungary.
authors: Pal
authors: P
authors: Boto
authors: Stem Cell Differentiation Laboratory, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen H-4010, Hungary.
authors: Renáta Nóra
authors: RN
authors: Szabó
authors: Stem Cell Differentiation Laboratory, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen H-4010, Hungary.
authors: Bianka Monika
authors: BM
authors: Toth
authors: Stem Cell Differentiation Laboratory, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen H-4010, Hungary.
authors: Emilia
authors: E
authors: Simo
authors: Genomic Medicine and Bioinformatic Core Facility, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen H-4010, Hungary.
authors: Bálint László
authors: BL
authors: Bálint
authors: Stem Cell Differentiation Laboratory, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen H-4010, Hungary; Faculty of Pharmacy, University of Debrecen, Debrecen H-4032, Hungary. Electronic address: szatmari@med.unideb.hu.
authors: Istvan
authors: I
authors: Szatmari
mesh_terms: D000107:Acetylation / Q000187:drug effects; D000818:Animals; D020148:Butyric Acid / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D019175:DNA Methylation / Q000187:drug effects; D004318:Doxycycline / Q000494:pharmacology*; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D019076:Transgenes / Q000235:genetics*; D000097573:Red Fluorescent Protein
chemical_list: D020148:Butyric Acid; D004318:Doxycycline; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein
keywords: Inducible cells; Mouse embryonic stem cells; Reporter genes; Transgenic cells; mCherry