title: 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.
authors.affiliation: Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: John
authors.initials: J
authors.lastname: Ramunas
authors.forename: Heather J
authors.initials: HJ
authors.lastname: Montgomery
authors.forename: Liam
authors.initials: L
authors.lastname: Kelly
authors.forename: Tanya
authors.initials: T
authors.lastname: Sukonnik
authors.forename: James
authors.initials: J
authors.lastname: Ellis
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Jervis
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D044127:Epigenesis, Genetic; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D011994:Recombinant Proteins / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D001374:Azacitidine