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Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

Lo MY, Rival-Gervier S, Pasceri P, Ellis J - PLoS ONE (2012)

Bottom Line: These transcription foci colocalized with the transgene integration site detected by immunoFISH.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.

View Article: PubMed Central - PubMed

Affiliation: Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Canada.

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.

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Detection of rapid transcriptional pulsing in Clone B6.A. Rapid and long transcriptional pulses (arrow) detected in a representative cell of Clone B6. Image acquisition was performed every 27 seconds. Scale bar = 2.5 µM. B. Intensity time series data (blue line) of the pulsing cell depicted in Figure 5A. Red line represents inferred transcriptional activity by visual scoring.
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pone-0037130-g005: Detection of rapid transcriptional pulsing in Clone B6.A. Rapid and long transcriptional pulses (arrow) detected in a representative cell of Clone B6. Image acquisition was performed every 27 seconds. Scale bar = 2.5 µM. B. Intensity time series data (blue line) of the pulsing cell depicted in Figure 5A. Red line represents inferred transcriptional activity by visual scoring.

Mentions: To determine whether rapid transcriptional pulsing events can be detected from other integration sites, we performed imaging studies on Clone B6. Because ES cell colonies have different thicknesses but were imaged in constant 300 nm z-dimension slices, each colony has a different number of sections in the z-stack and the thinner colonies can be captured more quickly than thicker ones. Therefore to minimize the image collection time during live imaging experiments, and taking into account the differences in the thickness of the ES cell colonies (12.0 µm to 18.2 µm), the imaging stack was acquired over 25 to 32 seconds (Video S2) for each timepoint. We detected rapid pulses of gene activity or inactivity that persisted only for one frame (ie. 25 to 32 seconds), but also longer pulses that remained for multiple frames as observed from the images (Figure 5A) and from the visual scoring (Figure 5B). We conclude transcriptional pulsing can be detected at two independent retroviral integration sites.


Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

Lo MY, Rival-Gervier S, Pasceri P, Ellis J - PLoS ONE (2012)

Detection of rapid transcriptional pulsing in Clone B6.A. Rapid and long transcriptional pulses (arrow) detected in a representative cell of Clone B6. Image acquisition was performed every 27 seconds. Scale bar = 2.5 µM. B. Intensity time series data (blue line) of the pulsing cell depicted in Figure 5A. Red line represents inferred transcriptional activity by visual scoring.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351450&req=5

pone-0037130-g005: Detection of rapid transcriptional pulsing in Clone B6.A. Rapid and long transcriptional pulses (arrow) detected in a representative cell of Clone B6. Image acquisition was performed every 27 seconds. Scale bar = 2.5 µM. B. Intensity time series data (blue line) of the pulsing cell depicted in Figure 5A. Red line represents inferred transcriptional activity by visual scoring.
Mentions: To determine whether rapid transcriptional pulsing events can be detected from other integration sites, we performed imaging studies on Clone B6. Because ES cell colonies have different thicknesses but were imaged in constant 300 nm z-dimension slices, each colony has a different number of sections in the z-stack and the thinner colonies can be captured more quickly than thicker ones. Therefore to minimize the image collection time during live imaging experiments, and taking into account the differences in the thickness of the ES cell colonies (12.0 µm to 18.2 µm), the imaging stack was acquired over 25 to 32 seconds (Video S2) for each timepoint. We detected rapid pulses of gene activity or inactivity that persisted only for one frame (ie. 25 to 32 seconds), but also longer pulses that remained for multiple frames as observed from the images (Figure 5A) and from the visual scoring (Figure 5B). We conclude transcriptional pulsing can be detected at two independent retroviral integration sites.

Bottom Line: These transcription foci colocalized with the transgene integration site detected by immunoFISH.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.

View Article: PubMed Central - PubMed

Affiliation: Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Canada.

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.

Show MeSH
Related in: MedlinePlus