title: Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.
abstract: Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES) cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.
authors.affiliation: Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Canada.
authors.forename: Mandy Y M
authors.initials: MY
authors.lastname: Lo
authors.forename: Sylvie
authors.initials: S
authors.lastname: Rival-Gervier
authors.forename: Peter
authors.initials: P
authors.lastname: Pasceri
authors.forename: James
authors.initials: J
authors.lastname: Ellis
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002842:Chromatids / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism* / Q000821:virology*; D015870:Gene Expression; D005814:Genes, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D017909:Levivirus / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012190:Retroviridae / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D019076:Transgenes; D016662:Virus Integration / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein