Real-time Fluorescence Tracking of Dynamic Transgene Variegation in Stem Cells

Abstract

Transgene variegation is caused by epigenetic switching between expressing and silent states. gamma-retrovirus vectors can be variegated in stem cells, but the dynamics of epigenetic remodeling during transgene variegation are unknown. Here, we measured variegated enhanced green fluorescent protein gamma-retrovirus expression over 4 days in individual embryonic stem cells while tracking cells in order to create expression lineage trees: 56 colony founder cells and their progeny were tracked over seven generations. Nineteen lineages produced synchronized inheritable trajectories of transgene silencing or reactivation, indicative of epigenetic remodeling with long-term stable inheritance. Short-term fluctuations in fluorescence intensity were also observed, which contributed low-amplitude variation to transgene expression level. These two processes have different frequencies and inheritability, but together contribute to variegated transgene expression. Inhibition of DNA methylation with 5-azacytidine eliminated long-term transgene silencing over 4 days, but short-term fluctuations continued. Our approach applies real-time imaging technology to track the long-term dynamics of transgene expression to investigate the timing and expression patterns leading to variegation.

Intraclonal Protein Expression Heterogeneity in Recombinant CHO Cells

“…We take an alternate approach, exploring the degree of variation within a clone, using single cell analysis facilitated by IRES-driven coexpression of intracellular fluorescent markers. Clones are normally assumed to be homogeneous but emerging fundamental research in bacteria [25]–[28], yeast [29]–[34], and more recently mammalian cells [35]–[39], has revealed that gene expression can vary significantly between genetically-identical cells, even in a common environment (reviewed in [40], [41]). We reasoned that this ‘hidden’ source of variation within clones [42], [43] might also have practical implications for cell line development, which are not yet widely appreciated.…”

Design and analysis of a long‐term live‐cell imaging chamber for tracking cellular dynamics within cultured human islets of Langerhans

“…1B). Previous studies have utilized the imaging chamber to restrict neurosphere growth to a two-dimensional monolayer and facilitate time course imaging and lineage informatics-based analysis (Ramunas et al, 2007). Although compression of an islet under the imaging chamber inevitably alters its structure, it was thought that cell-cell and cell-extracellular matrix connectivity and overall islet cell functions could be maintained by appropriate selection of both initial islet size and imaging chamber gap spacing.…”

“…The imaging chamber described herein has previously been implemented to study monolayer cultures (Karpowicz et al, 2005;Ramunas et al, 2007). Herein, chamber design criteria were investigated to ensure the imaging chamber could be used for collagen-embedded three dimensional cultures of human islets of Langerhans.…”

A novel view on stem cell development: analysing the shape of cellular genealogies

“…It should be noted that this approach can also be generalized to other events that characterize the fate of sibling cells. A related but less analytical approach to correlate fluorescence expression between closely related cells, indicating synchronized epigenetic remodelling in embryonic stem cells, has been published recently (10).…”