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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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Related in: MedlinePlus

Development of the MS2 system to detect transcription sites of a retroviral transgene.A. Schematic diagram for detecting transcription sites from retroviral transgenes. The MS2 stem-loop was inserted into a HSC1 retrovirus backbone expressing mRFP. The structure of provirus after integration into the genome is shown. Location of restriction enzymes digestion sites, probe used for southern blot analysis (gray box) and primers for PCR (red arrows) to confirm size of stem-loops are indicated. B. Southern blot analysis of genomic DNA from Clone B6 and 3A10 of infected ES cells digested with various enzymes. (E = EcoRI, B = BamHI, H = HindIII, S = SpeI, N = NheI) Digested DNA was hybridized to the mRFP probe. No HindIII site is found within the provirus. C. PCR analysis of number of stem-loops integrated into the genome in the two clones (top) and the number of stem-loops transcribed from each clone (bottom). Uninfected J1 ES cells were used as a control. Actin was used as a loading control. D. Flow cytometry histograms showing expression of mRFP in Clone B6 (top) and Clone 3A10 (bottom). Red line denotes uninfected control J1 cells and blue line indicates clone being interrogated.
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pone-0037130-g001: Development of the MS2 system to detect transcription sites of a retroviral transgene.A. Schematic diagram for detecting transcription sites from retroviral transgenes. The MS2 stem-loop was inserted into a HSC1 retrovirus backbone expressing mRFP. The structure of provirus after integration into the genome is shown. Location of restriction enzymes digestion sites, probe used for southern blot analysis (gray box) and primers for PCR (red arrows) to confirm size of stem-loops are indicated. B. Southern blot analysis of genomic DNA from Clone B6 and 3A10 of infected ES cells digested with various enzymes. (E = EcoRI, B = BamHI, H = HindIII, S = SpeI, N = NheI) Digested DNA was hybridized to the mRFP probe. No HindIII site is found within the provirus. C. PCR analysis of number of stem-loops integrated into the genome in the two clones (top) and the number of stem-loops transcribed from each clone (bottom). Uninfected J1 ES cells were used as a control. Actin was used as a loading control. D. Flow cytometry histograms showing expression of mRFP in Clone B6 (top) and Clone 3A10 (bottom). Red line denotes uninfected control J1 cells and blue line indicates clone being interrogated.

Mentions: In order to determine transcriptional dynamics of retroviral transgenes in ES cells we employed an HSC1-EF1α-mRFP-MS2 vector design. A self-inactivating HSC1 retrovirus backbone with an ubiquitous elongation factor-1α (EF1α) promoter driving the expression of mRFP and the MS2 stem-loop cassette (Figure 1A) was generated. We infected J1 mouse ES cells with this virus at low multiplicity of infection (<0.6) to identify single copy integrants expressing the mRFP-MS2 retrovirus. Two expressor cell lines were isolated through cell sorting for mRFP expression from two independent infections. To confirm both cell lines contained retrovirus inserted as a single copy integrant, southern blot analysis was performed using a probe hybridizing to the mRFP gene. The restriction enzymes EcoRI or BamHI cleave the integrated provirus once, and only one band was observed for both clones (Figure 1B). Digestion with HindIII, which is not found on the integrated provirus, also yields a single band of different size for both clones, confirming the two proviruses are integrated at different integration sites. Digestion with NheI and SpeI also revealed that the MS2 stem-loop fragment was longer in Clone B6 than Clone 3A10 (Figure 1B). To further confirm this result, PCR was performed on genomic DNA of both clones to examine the number of MS2-stemloops that were successfully transmitted through the retrovirus and integrated into the genome of the clones. A full set of 24 MS2 stem-loops would yield a band of 1.4 kb. The size of the amplicon was 1.4 kb from Clone B6 and 0.9 kb from Clone 3A10, which corresponds to 24 stem-loops and 16 stem-loops respectively (Figure 1C). Since direct repeat sequences are difficult to reverse transcribe intact through retroviral vectors, the reduction of stem-loops in Clone 3A10 is not unexpected [18]. RT-PCR analysis shows that the same number of stem-loops was transcribed into RNA (Figure 1C). Flow cytometry analysis was performed to observe the expression of the provirus in these two lines. Both Clone B6 and Clone 3A10 have >98% mRFP+ cells that express to high levels (MFI = 3523 and 2544 respectively), although Clone 3A10 displayed a wider peak (CV = 94.1) compared to Clone B6 (CV = 60.2), suggesting more cell-to-cell variability in mRFP expression levels (Figure 1D). Such high levels of expression are compatible with detection of transcription foci using the MS2 system.


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)

Development of the MS2 system to detect transcription sites of a retroviral transgene.A. Schematic diagram for detecting transcription sites from retroviral transgenes. The MS2 stem-loop was inserted into a HSC1 retrovirus backbone expressing mRFP. The structure of provirus after integration into the genome is shown. Location of restriction enzymes digestion sites, probe used for southern blot analysis (gray box) and primers for PCR (red arrows) to confirm size of stem-loops are indicated. B. Southern blot analysis of genomic DNA from Clone B6 and 3A10 of infected ES cells digested with various enzymes. (E = EcoRI, B = BamHI, H = HindIII, S = SpeI, N = NheI) Digested DNA was hybridized to the mRFP probe. No HindIII site is found within the provirus. C. PCR analysis of number of stem-loops integrated into the genome in the two clones (top) and the number of stem-loops transcribed from each clone (bottom). Uninfected J1 ES cells were used as a control. Actin was used as a loading control. D. Flow cytometry histograms showing expression of mRFP in Clone B6 (top) and Clone 3A10 (bottom). Red line denotes uninfected control J1 cells and blue line indicates clone being interrogated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351450&req=5

pone-0037130-g001: Development of the MS2 system to detect transcription sites of a retroviral transgene.A. Schematic diagram for detecting transcription sites from retroviral transgenes. The MS2 stem-loop was inserted into a HSC1 retrovirus backbone expressing mRFP. The structure of provirus after integration into the genome is shown. Location of restriction enzymes digestion sites, probe used for southern blot analysis (gray box) and primers for PCR (red arrows) to confirm size of stem-loops are indicated. B. Southern blot analysis of genomic DNA from Clone B6 and 3A10 of infected ES cells digested with various enzymes. (E = EcoRI, B = BamHI, H = HindIII, S = SpeI, N = NheI) Digested DNA was hybridized to the mRFP probe. No HindIII site is found within the provirus. C. PCR analysis of number of stem-loops integrated into the genome in the two clones (top) and the number of stem-loops transcribed from each clone (bottom). Uninfected J1 ES cells were used as a control. Actin was used as a loading control. D. Flow cytometry histograms showing expression of mRFP in Clone B6 (top) and Clone 3A10 (bottom). Red line denotes uninfected control J1 cells and blue line indicates clone being interrogated.
Mentions: In order to determine transcriptional dynamics of retroviral transgenes in ES cells we employed an HSC1-EF1α-mRFP-MS2 vector design. A self-inactivating HSC1 retrovirus backbone with an ubiquitous elongation factor-1α (EF1α) promoter driving the expression of mRFP and the MS2 stem-loop cassette (Figure 1A) was generated. We infected J1 mouse ES cells with this virus at low multiplicity of infection (<0.6) to identify single copy integrants expressing the mRFP-MS2 retrovirus. Two expressor cell lines were isolated through cell sorting for mRFP expression from two independent infections. To confirm both cell lines contained retrovirus inserted as a single copy integrant, southern blot analysis was performed using a probe hybridizing to the mRFP gene. The restriction enzymes EcoRI or BamHI cleave the integrated provirus once, and only one band was observed for both clones (Figure 1B). Digestion with HindIII, which is not found on the integrated provirus, also yields a single band of different size for both clones, confirming the two proviruses are integrated at different integration sites. Digestion with NheI and SpeI also revealed that the MS2 stem-loop fragment was longer in Clone B6 than Clone 3A10 (Figure 1B). To further confirm this result, PCR was performed on genomic DNA of both clones to examine the number of MS2-stemloops that were successfully transmitted through the retrovirus and integrated into the genome of the clones. A full set of 24 MS2 stem-loops would yield a band of 1.4 kb. The size of the amplicon was 1.4 kb from Clone B6 and 0.9 kb from Clone 3A10, which corresponds to 24 stem-loops and 16 stem-loops respectively (Figure 1C). Since direct repeat sequences are difficult to reverse transcribe intact through retroviral vectors, the reduction of stem-loops in Clone 3A10 is not unexpected [18]. RT-PCR analysis shows that the same number of stem-loops was transcribed into RNA (Figure 1C). Flow cytometry analysis was performed to observe the expression of the provirus in these two lines. Both Clone B6 and Clone 3A10 have >98% mRFP+ cells that express to high levels (MFI = 3523 and 2544 respectively), although Clone 3A10 displayed a wider peak (CV = 94.1) compared to Clone B6 (CV = 60.2), suggesting more cell-to-cell variability in mRFP expression levels (Figure 1D). Such high levels of expression are compatible with detection of transcription foci using the MS2 system.

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