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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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Nuclear positioning of transcriptional foci.A. ImmunoFISH of Clone B6 (top) and Clone 3A10 (bottom) with their respective BAC probes (red) against the integration site, and immunofluorescence staining with EGFP antibodies (green) and DAPI (cyan). A single focal plane with the focal dot is shown. B. ImmunoFISH of Clone B6 cells showing an EGFP focal dot (green) located at RNA Pol II factories (violet) and DAPI (cyan). A single focal plane with the focal dot is shown. Scale bar = 2.5 µM. C. Representative image of Dlgap4 loci (red) in Clone B6 with respect to the nuclear periphery (violet). The nucleus is counterstained by DAPI (cyan). Scale bar = 2.5 µM. Image shown is the maximal projection of multiple stacks. D. Quantification of the percentage of BAC FISH signals marking the Clone B6 integration site found at the nuclear periphery compared to J1 cells (n = 3, 104–140 FISH signals were counted for each n). E. Quantification of the percentage of BAC FISH signals marking the Clone 3A10 integration site found at the nuclear periphery compared to J1 cells (n = 4, 106–134 FISH signals were counted for each n).
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pone-0037130-g003: Nuclear positioning of transcriptional foci.A. ImmunoFISH of Clone B6 (top) and Clone 3A10 (bottom) with their respective BAC probes (red) against the integration site, and immunofluorescence staining with EGFP antibodies (green) and DAPI (cyan). A single focal plane with the focal dot is shown. B. ImmunoFISH of Clone B6 cells showing an EGFP focal dot (green) located at RNA Pol II factories (violet) and DAPI (cyan). A single focal plane with the focal dot is shown. Scale bar = 2.5 µM. C. Representative image of Dlgap4 loci (red) in Clone B6 with respect to the nuclear periphery (violet). The nucleus is counterstained by DAPI (cyan). Scale bar = 2.5 µM. Image shown is the maximal projection of multiple stacks. D. Quantification of the percentage of BAC FISH signals marking the Clone B6 integration site found at the nuclear periphery compared to J1 cells (n = 3, 104–140 FISH signals were counted for each n). E. Quantification of the percentage of BAC FISH signals marking the Clone 3A10 integration site found at the nuclear periphery compared to J1 cells (n = 4, 106–134 FISH signals were counted for each n).

Mentions: To confirm the integration sites and that the green focal dots observed previously are indeed sites of transgene transcription, a BAC DNA-FISH probe that spans the Dlgap4 integration site was labeled and immunoFISH performed on Clone B6 using an antibody against EGFP. Transcription foci were marked by EGFP accumulation in a focal dot and were found to colocalize to only one allele of the genomic integration site as expected (Figure 3A). This analysis verifies that the LM-PCR result identified the correct integration site and that the focal dots specifically mark the provirus. A similar result was obtained when DNA FISH was performed on Clone 3A10 using a BAC probe against its integration site (Figure 3A).


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)

Nuclear positioning of transcriptional foci.A. ImmunoFISH of Clone B6 (top) and Clone 3A10 (bottom) with their respective BAC probes (red) against the integration site, and immunofluorescence staining with EGFP antibodies (green) and DAPI (cyan). A single focal plane with the focal dot is shown. B. ImmunoFISH of Clone B6 cells showing an EGFP focal dot (green) located at RNA Pol II factories (violet) and DAPI (cyan). A single focal plane with the focal dot is shown. Scale bar = 2.5 µM. C. Representative image of Dlgap4 loci (red) in Clone B6 with respect to the nuclear periphery (violet). The nucleus is counterstained by DAPI (cyan). Scale bar = 2.5 µM. Image shown is the maximal projection of multiple stacks. D. Quantification of the percentage of BAC FISH signals marking the Clone B6 integration site found at the nuclear periphery compared to J1 cells (n = 3, 104–140 FISH signals were counted for each n). E. Quantification of the percentage of BAC FISH signals marking the Clone 3A10 integration site found at the nuclear periphery compared to J1 cells (n = 4, 106–134 FISH signals were counted for each n).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351450&req=5

pone-0037130-g003: Nuclear positioning of transcriptional foci.A. ImmunoFISH of Clone B6 (top) and Clone 3A10 (bottom) with their respective BAC probes (red) against the integration site, and immunofluorescence staining with EGFP antibodies (green) and DAPI (cyan). A single focal plane with the focal dot is shown. B. ImmunoFISH of Clone B6 cells showing an EGFP focal dot (green) located at RNA Pol II factories (violet) and DAPI (cyan). A single focal plane with the focal dot is shown. Scale bar = 2.5 µM. C. Representative image of Dlgap4 loci (red) in Clone B6 with respect to the nuclear periphery (violet). The nucleus is counterstained by DAPI (cyan). Scale bar = 2.5 µM. Image shown is the maximal projection of multiple stacks. D. Quantification of the percentage of BAC FISH signals marking the Clone B6 integration site found at the nuclear periphery compared to J1 cells (n = 3, 104–140 FISH signals were counted for each n). E. Quantification of the percentage of BAC FISH signals marking the Clone 3A10 integration site found at the nuclear periphery compared to J1 cells (n = 4, 106–134 FISH signals were counted for each n).
Mentions: To confirm the integration sites and that the green focal dots observed previously are indeed sites of transgene transcription, a BAC DNA-FISH probe that spans the Dlgap4 integration site was labeled and immunoFISH performed on Clone B6 using an antibody against EGFP. Transcription foci were marked by EGFP accumulation in a focal dot and were found to colocalize to only one allele of the genomic integration site as expected (Figure 3A). This analysis verifies that the LM-PCR result identified the correct integration site and that the focal dots specifically mark the provirus. A similar result was obtained when DNA FISH was performed on Clone 3A10 using a BAC probe against its integration site (Figure 3A).

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