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.

Rosa26 docking sites for investigating genetic circuit silencing in stem cells

“…EGFP was assessed using microscopy and results show that ES colonies that are cultured beyond three weeks significantly lose their ability to express EGFP in the presence of IPTG compared to cells in earlier time points ( Figure 3C-D'). Altogether, these data suggest that the decrease in gene expression strength is likely due to the genetic circuit being susceptible to epigenetic modifications, specifically to methylation and/or histone acetylation (33,34,58).…”

“…These model cell lines are useful for enhancing our understanding of synthetic gene circuits that underscore the potential of synthetic biology tools, however, these cell lines may not be good predictors of the challenges that arise in stem cells (31). Plasmid and viral gene delivery systems have shown to lose expression over weeks of cell culture, which is thought to be a consequence of epigenetic modifications of the inserted DNA (32)(33)(34). The current understanding of transgene silencing suggests that silencing of genes can occur through the methylation of the expression cassette and/or the formation of heterochromatin, both of which facilitate changes in gene expression; however, the circumstances that trigger these mechanisms are still being elucidated.…”

Immunogenic Dendritic Cell Generation from Pluripotent Stem Cells by Ectopic Expression of Runx3

“…The Runx3-carrying Gateway master vector (pENTR223.1; clone ID: FLH481113.01X) was purchased from the DNASU plasmid depository. The obtained Gateway entry clones were recombined to a modified p2Lox plasmid (17,18) containing Gateway destination sequences using the Gateway Cloning System (Life Technologies). For ES cell transfection, 5 mg of the targeting constructs was electroporated into ZX1 (19) ES cells with the Neon Transfection System (Life Technologies).…”

“…For ES cell transfection, 5 mg of the targeting constructs was electroporated into ZX1 (19) ES cells with the Neon Transfection System (Life Technologies). Inducible cassette-exchange recombination was used to insert the selected open reading frames into a euchromatic site on the X chromosome (17,18). ES cell colonies were selected in 300 mg/ml G418 containing ES cell medium, picked on day 8, and expanded.…”

“…In light of this altered gene expression, we probed the effects of the underexpressed transcription factors (Spi-B, Irf4, and Runx3) in developing ES-DCs using isogenic expression screening. We applied a genetically modified mouse ES cell line (ZX1), into which a gene of interest can be inserted by Cre-mediated recombination (17)(18)(19). In these cells, the investigated genes can be induced in response to tetracycline or doxycycline (Tet-on system).…”