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• W1967823954 abstract "The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can stably integrate into the genomes of mammalian cells. Although SB is one of the most efficient non-viral integrating systems described to date, all previous estimations of its integrative potential have relied on the use of antibiotic selection schemes. Based on previous studies demonstrating post-integrative gene silencing with other gene transfer systems (e.g. retroviruses), we investigated how genomic integration influenced transposon expression in human cells. To do this, we devised a novel strategy to monitor SB integration irrespective of transgene expression. We first constructed single plasmids (pT/RSV- YFP.CMV-SB, pT/EF1α-eGFP.CMV-SB, and pT/dmEF1α- dmGFP.CMV-SB) encoding a GFP variant-marked transposon and an externally-located transposase expression cassette and then transfected each of these plasmids into HeLa cells. Shortly thereafter, cells were single-cell sorted by FACS and then grown in the absence of antibiotic selection to generate a large number of clonal cell lines, each of which was screened via Southern blot analysis for transposon integrations. Results indicate that in the absence of selection, the Sleeping Beauty transposition system has a true integration efficiency of 41-52%, a value that is significantly higher than the 3-10% estimates previously generated using antibiotic selection-based approaches. In addition, we find that, irrespective of promoter usage or CpG content, many transposon copies undergo post-integrational gene silencing during a 12 week time period, with silencing in 13-18% of single-integration cell lines. Furthermore, upon continued passage of the pT/RSV-YFP.CMV-SB-derived lines, the frequency of transposon silencing had increased substantially from 18% to 71% after a 42 week time period. Studies utilizing the methylation- sensitive restriction enzyme McrBC indicate that DNA methylation contributes substantially to transgene repression in the vast majority of integrated transposon copies, a finding which was further substantiated through experimental application of the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine. Histone deacetylation was also found to play a role in transgene repression using the histone deacetylases inhibitor trichostatin A. Overall, these transposon clones are proving invaluable in helping unravel the molecular mechanism(s) governing transcriptional gene silencing, and may someday provide the insight needed to design an appropriate integrating vector system that is capable of maintaining long-term gene expression regardless of its integration site. The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can stably integrate into the genomes of mammalian cells. Although SB is one of the most efficient non-viral integrating systems described to date, all previous estimations of its integrative potential have relied on the use of antibiotic selection schemes. Based on previous studies demonstrating post-integrative gene silencing with other gene transfer systems (e.g. retroviruses), we investigated how genomic integration influenced transposon expression in human cells. To do this, we devised a novel strategy to monitor SB integration irrespective of transgene expression. We first constructed single plasmids (pT/RSV- YFP.CMV-SB, pT/EF1α-eGFP.CMV-SB, and pT/dmEF1α- dmGFP.CMV-SB) encoding a GFP variant-marked transposon and an externally-located transposase expression cassette and then transfected each of these plasmids into HeLa cells. Shortly thereafter, cells were single-cell sorted by FACS and then grown in the absence of antibiotic selection to generate a large number of clonal cell lines, each of which was screened via Southern blot analysis for transposon integrations. Results indicate that in the absence of selection, the Sleeping Beauty transposition system has a true integration efficiency of 41-52%, a value that is significantly higher than the 3-10% estimates previously generated using antibiotic selection-based approaches. In addition, we find that, irrespective of promoter usage or CpG content, many transposon copies undergo post-integrational gene silencing during a 12 week time period, with silencing in 13-18% of single-integration cell lines. Furthermore, upon continued passage of the pT/RSV-YFP.CMV-SB-derived lines, the frequency of transposon silencing had increased substantially from 18% to 71% after a 42 week time period. Studies utilizing the methylation- sensitive restriction enzyme McrBC indicate that DNA methylation contributes substantially to transgene repression in the vast majority of integrated transposon copies, a finding which was further substantiated through experimental application of the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine. Histone deacetylation was also found to play a role in transgene repression using the histone deacetylases inhibitor trichostatin A. Overall, these transposon clones are proving invaluable in helping unravel the molecular mechanism(s) governing transcriptional gene silencing, and may someday provide the insight needed to design an appropriate integrating vector system that is capable of maintaining long-term gene expression regardless of its integration site." @default.
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• W1967823954 date "2006-01-01" @default.
• W1967823954 modified "2023-09-26" @default.
• W1967823954 title "792. Post-Integrative Gene Silencing in the Sleeping Beauty Transposition System" @default.
• W1967823954 doi "https://doi.org/10.1016/j.ymthe.2006.08.880" @default.
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