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.