We previously demonstrated that plasmid bacterial DNA sequences transcriptionally inactivate transgene expression while minicircle DNA vector free of plasmid bacterial backbone is persistently active in vivo. To further elucidate the underlying molecular mechanism, we studied the conditions required for the plasmid bacterial backbone to suppress transgene expression. We found that when circular or linearized minicircle encoding the RSV.hAAT.bpA expression cassette co-injected with circular or linear pBlueScript to mouse liver via hydrodynamic technique, transgene silencing occurred when 2 linear DNAs were infused together, but not when either one or both DNA elements were in circular form, or if the linear expression cassette was injected alone. Southern analyses confirmed that linear DNA but not circular DNA molecules formed random concatemers. This further confirmed our earlier findings that transcriptional silencing occurred only when the bacterial plasmid sequences were covalently attached to the expression cassette, and bacterium-derived plasmid DNA molecules, either in circular or in concatemer form, present in the same cells will not silence DNA expression cassettes in trans-. Furthermore, we found that after injection of a mixture of linear DNA expression cassettes and plasmid bacterial DNAs into normal or SCID mice, silencing was observed in both strains of mouse. These observations strongly suggest that toxic, and probably immunogenic, signaling from plasmid derived DNAs is not responsible for DNA silencing. To determine if DNA methylation was associated with silencing, both transgene and bacterial backbone DNAs were generated from the cytosine methylation-deficient bacterial strain, SCS110. Southern analyses using methylation sensitive restriction enzymes, showed no in vivo cytosine methylation in the CpG motif. This suggests that DNA methylation is not responsible for transcriptional silencing. Interestingly, transgene silencing was alleviated when the RSV.hAAT.bpA expression cassette was flanked with 2 tandem copies of the chicken SH4 insulator at each end. Taken together our results support our hypothesis that the transcriptional suppression is due to differential nucleosome formation and/or histone modifications that are formed in the presence of plasmid bacterial DNA sequences, and then spread to suppress the transgene promoter in cis-.