| 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 the most efficient non-viral integrating system described to date, all previous estimations of SB's integrative potential have relied on the use of antibiotic selection schemes. Previous work in other integrating systems (e.g. retroviruses) has demonstrated significant post-integrative gene silencing, leading us to investigate how genomic integration influences transposon expression in human cells. To do this we devised a novel strategy to monitor SB integration that is not dependent on transgene expression. We constructed single plasmids (pT/RSV-YFP.CMV-SB, pT/EF1alpha-eGFP.CMV-SB, and pT/dmEF1alpha-dmGFP.CMV-SB) containing both a GFP variant-marked transposon and a separate transposase expression cassette (external to the transposon), then transfected the plasmids into HeLa cells. Shortly thereafter, cells were single-cell sorted by FACS and grown in the absence of antibiotic selection to generate clonal cell lines, which were then screened via Southern blot analysis for transposon integrations. Results indicate that in the absence of selection the Sleeping Beauty transposition system has an integration efficiency of 41-52%, which is much higher than the previously reported 3-10% obtained using antibiotic selection approaches. In addition, our results demonstrate that many transposons experience post-integrational silencing since at week 12 our vectors had been silenced in 13-18% of the integration-containing cell lines. Furthermore, the cell lines created using pT/RSV-YFP.CMV-SB vector were passaged long-term to week 42 at which point silencing within that data set had increased from 18% to 71%. Recent data using the methylation sensitive restriction enzyme McrBC indicates DNA methylation plays a role in transgene repression within almost all the integrated transposons, which was verified by experiments using the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine. Histone deacetylation was also shown to play a role in transgene repression using the histone deacetylases inhibitor trichostatin A. Overall, our transposon clones are proving to be an important new reagent to investigate the mechanism(s) by which integrating genetic elements (e.g. transposons and retroviruses) are silenced by the host cell, and should further our understanding of transcriptional silencing of integrating gene transfer vectors. |
