The deletion of a selection cassette lying between the endogenous ROSA26 promoter and a CMV driven rtTA expression cassette reduced the activity of the transgene, suggesting that this selection cassette was previously screening the transgene from these interference effects to a certain extent. Interestingly, the significant orientation dependent effects seen in this study suggest a trend towards increased expression in the sense orientation. However, the construct AG-013736 arrangement in this study includes the positioning of a mouse H19 insulator element between the expression cassette and the endogenous ROSA26 promoter and, subsequently, it is likely that this serves to block all transcriptional read-through from the ROSA26 promoter which may otherwise contribute to transcriptional interference with exogenous promoters positioned in the sense orientation. Indeed, the insertion of the promoterless luciferase cassettes in the sense orientation led to no discernible expression. Read-through transcription in the antisense orientation is not considered to influence the expression of transgenes inserted using his system as the recognised antisense transcript terminates 8 kb downstream of the transgene insertion site. In agreement, ES cell clones in which a promoterless luciferase cassette was positioned in the antisense orientation revealed only background levels of activity. Despite the apparent lack of transcriptional read-through activity from the ROSA26 promoter into the inserted transgenes in this system, significant orientation dependent effects were observed for the EF1a promoter with borderline significant effects being recorded for the MC1 and the UbC promoters. The results of the transient transfection assay, however, revealed no orientation dependent effects for these promoters, suggesting that these differences were not a consequence of transgene arrangement with respect to the plasmid backbone, the selection cassette, the insulator or the integration machinery. Instead, these effects must be dependent upon integration within the ROSA26 locus and thus potentially reflect the accessibility of these promoter elements for transcription factors or steric considerations. In conclusion, the results presented in this study quantify the strengths of commonly used ubiquitous promoters when integrated at single copy within the ROSA26 locus. The study also serves to validate the PhiC31 integrase mediated cassette exchange approach for the analysis of multiple variant constructs and demonstrates the relative ease and robustness of performing multiple comparative analyses.