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DNA methylation and histone modifications are the molecular lock in lentivirally transduced hematopoietic progenitor cells.

Ngai SC, Rosli R, Al Abbar A, Abdullah S - Biomed Res Int (2015)

Bottom Line: Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect.When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state.With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV.

View Article: PubMed Central - PubMed

Affiliation: Medical Genetics Laboratory, Clinical Genetics Unit, Faculty of Medicine and Health Sciences, UPM, 43400 Serdang, Selangor, Malaysia ; School of Biosciences, Faculty of Science, The University of Nottingham Malaysia, Jalan Broga, 43500 Semenyih, Selangor, Malaysia.

ABSTRACT
Stable introduction of a functional gene in hematopoietic progenitor cells (HPCs) has appeared to be an alternative approach to correct genetically linked blood diseases. However, it is still unclear whether lentiviral vector (LV) is subjected to gene silencing in HPCs. Here, we show that LV carrying green fluorescent protein (GFP) reporter gene driven by cytomegalovirus (CMV) promoter was subjected to transgene silencing after transduction into HPCs. This phenomenon was not due to the deletion of proviral copy number. Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect. Using sodium bisulfite sequencing and chromatin immunoprecipitation (ChIP) assay, we demonstrated that DNA methylation occurred soon after LV transduction. At the highest level of gene expression, CMV promoter was acetylated and was in a euchromatin state, while GFP reporter gene was acetylated but was strangely in a heterochromatin state. When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state. With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV.

No MeSH data available.


Related in: MedlinePlus

Relative transgene copy number. The extracted genomic DNA from the transduced cells at day 2 and 7 after transduction was subjected to PCR to amplify the transgene region of the sample. Lane 1: 1 kb ladder; Lanes 2–4: PCR with primers specific for HPRT gene; Lane 2: Untransduced genomic DNA (positive control for PCR); Lane 3: transduced genomic DNA extracted at day 2 after transduction (positive control for relative transgene copy number); Lane 4: transduced genomic DNA extracted at day 7 after transduction (positive control for relative transgene copy number); Lane 5: PCR reaction without DNA (negative control); Lane 6–9: PCR with primers specific to transgene; Lane 6: untransduced genomic DNA; Lane 7: transduced genomic DNA extracted at day 2 after transduction; Lane 8: transduced genomic DNA extracted at day 7 after transduction; Lane 9: PCR reaction without DNA (negative control for PCR).
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fig3: Relative transgene copy number. The extracted genomic DNA from the transduced cells at day 2 and 7 after transduction was subjected to PCR to amplify the transgene region of the sample. Lane 1: 1 kb ladder; Lanes 2–4: PCR with primers specific for HPRT gene; Lane 2: Untransduced genomic DNA (positive control for PCR); Lane 3: transduced genomic DNA extracted at day 2 after transduction (positive control for relative transgene copy number); Lane 4: transduced genomic DNA extracted at day 7 after transduction (positive control for relative transgene copy number); Lane 5: PCR reaction without DNA (negative control); Lane 6–9: PCR with primers specific to transgene; Lane 6: untransduced genomic DNA; Lane 7: transduced genomic DNA extracted at day 2 after transduction; Lane 8: transduced genomic DNA extracted at day 7 after transduction; Lane 9: PCR reaction without DNA (negative control for PCR).

Mentions: Figure 3 shows that the expected bands were obtained in Lanes 2, 3, and 4 which were set as the controls, indicating that the mouse housekeeping HPRT gene was expressed in both untransduced and transduced murine HPCs harvested at day 2 and day 7 after transduction. No band was obtained in Lane 5, which was set as negative control for PCR. For PCR with primers specific to the transgene, no band was obtained for the untransduced cells (Lane 6), indicating no integrated provirus in the cells. Expected bands were obtained for the LV-transduced cells harvested at day 2 and day 7 after transduction, indicating that the provirus was integrated into the cells genome. No band was obtained for PCR reaction mixture without DNA, which was set as negative control.


DNA methylation and histone modifications are the molecular lock in lentivirally transduced hematopoietic progenitor cells.

Ngai SC, Rosli R, Al Abbar A, Abdullah S - Biomed Res Int (2015)

Relative transgene copy number. The extracted genomic DNA from the transduced cells at day 2 and 7 after transduction was subjected to PCR to amplify the transgene region of the sample. Lane 1: 1 kb ladder; Lanes 2–4: PCR with primers specific for HPRT gene; Lane 2: Untransduced genomic DNA (positive control for PCR); Lane 3: transduced genomic DNA extracted at day 2 after transduction (positive control for relative transgene copy number); Lane 4: transduced genomic DNA extracted at day 7 after transduction (positive control for relative transgene copy number); Lane 5: PCR reaction without DNA (negative control); Lane 6–9: PCR with primers specific to transgene; Lane 6: untransduced genomic DNA; Lane 7: transduced genomic DNA extracted at day 2 after transduction; Lane 8: transduced genomic DNA extracted at day 7 after transduction; Lane 9: PCR reaction without DNA (negative control for PCR).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4417590&req=5

fig3: Relative transgene copy number. The extracted genomic DNA from the transduced cells at day 2 and 7 after transduction was subjected to PCR to amplify the transgene region of the sample. Lane 1: 1 kb ladder; Lanes 2–4: PCR with primers specific for HPRT gene; Lane 2: Untransduced genomic DNA (positive control for PCR); Lane 3: transduced genomic DNA extracted at day 2 after transduction (positive control for relative transgene copy number); Lane 4: transduced genomic DNA extracted at day 7 after transduction (positive control for relative transgene copy number); Lane 5: PCR reaction without DNA (negative control); Lane 6–9: PCR with primers specific to transgene; Lane 6: untransduced genomic DNA; Lane 7: transduced genomic DNA extracted at day 2 after transduction; Lane 8: transduced genomic DNA extracted at day 7 after transduction; Lane 9: PCR reaction without DNA (negative control for PCR).
Mentions: Figure 3 shows that the expected bands were obtained in Lanes 2, 3, and 4 which were set as the controls, indicating that the mouse housekeeping HPRT gene was expressed in both untransduced and transduced murine HPCs harvested at day 2 and day 7 after transduction. No band was obtained in Lane 5, which was set as negative control for PCR. For PCR with primers specific to the transgene, no band was obtained for the untransduced cells (Lane 6), indicating no integrated provirus in the cells. Expected bands were obtained for the LV-transduced cells harvested at day 2 and day 7 after transduction, indicating that the provirus was integrated into the cells genome. No band was obtained for PCR reaction mixture without DNA, which was set as negative control.

Bottom Line: Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect.When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state.With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV.

View Article: PubMed Central - PubMed

Affiliation: Medical Genetics Laboratory, Clinical Genetics Unit, Faculty of Medicine and Health Sciences, UPM, 43400 Serdang, Selangor, Malaysia ; School of Biosciences, Faculty of Science, The University of Nottingham Malaysia, Jalan Broga, 43500 Semenyih, Selangor, Malaysia.

ABSTRACT
Stable introduction of a functional gene in hematopoietic progenitor cells (HPCs) has appeared to be an alternative approach to correct genetically linked blood diseases. However, it is still unclear whether lentiviral vector (LV) is subjected to gene silencing in HPCs. Here, we show that LV carrying green fluorescent protein (GFP) reporter gene driven by cytomegalovirus (CMV) promoter was subjected to transgene silencing after transduction into HPCs. This phenomenon was not due to the deletion of proviral copy number. Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect. Using sodium bisulfite sequencing and chromatin immunoprecipitation (ChIP) assay, we demonstrated that DNA methylation occurred soon after LV transduction. At the highest level of gene expression, CMV promoter was acetylated and was in a euchromatin state, while GFP reporter gene was acetylated but was strangely in a heterochromatin state. When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state. With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV.

No MeSH data available.


Related in: MedlinePlus