The heterogeneity or mosaicism of stem cell CMV expression due to epigenetic silencing of randomly integrated transgenes is now well established and may explain some of the inconsistencies seen in the published studies. Of the vertebrate BYL719 1217486-61-7 promoters tested, EF1a yielded robust expression levels approximately 4.5 fold higher than the ROSA26 promoter when positioned in the sense orientation. Previous reports have also confirmed the moderate expression levels of EF1a in transient, stably transfected and adenoviral infected ES cell clones. In contrast to our results, a further study using lentiviral infection of mouse ES cells has suggested EF1a promoter strength to be approximately two fold higher than that of the CAG promoter. However, although the authors controlled for copy number integration by quantitative PCR, the multiplicity of random integration events which occurs during lentiviral infection could still be a source of variation in the experimental read-out. UbC promoter driven expression was found to be 3–5 fold higher than the endogenous ROSA26 promoter. Despite the well characterised ubiquitous expression observed in UbC driven transgenic mice, there is little data assessing the expression level of UbC in mouse ES cells. Strong transgene expression driven from UbC has however been confirmed in human stem cells and human hematopoietic and murine mesenchymal progenitor cells. PGK, chicken b-actin and MC1 promoter activity was found to be at a comparable level with that of the endogenous ROSA26 promoter. There is little previous evidence directly comparing the activities of these promoters, although one study has assessed the strength of these promoters for achieving Cre recombinase mediated cassette deletions. This indirect evidence of promoter strength concluded that PGK and MC1 promoter activity are significantly lower than that of CAG and EF1a, in agreement with this study. In terms of orientation dependent effects, two published studies have revealed that certain promoters behave differently if positioned in an antisense or sense orientation at the ROSA26 locus. CAG promoter driven transgene expression was found to be at least ten fold higher in the antisense orientation than in the sense orientation and CMV driven expression of the reverse tetracycline transactivator was inferred to be more robustly expressed in the antisense orientation. Transcriptional readthrough interference from the ROSA26 sense transcripts was considered to be responsible for the lower level of expression of transgenes positioned.