Douglas Strathdee, Helen Ibbotson and Seth G. N. Grant

“ Conclusions Expression of the targeted transgenes was shown to be affected by both the presence of selectable marker cassettes and by the orientation of the transgenes with respect to the endogenous ROSA26 promoter. These results suggest that transcriptional interference from the endogenous gene promoter or from promoters in the selectable marker cassettes may be affecting transgene expression at the locus. Additionally we have been able to determine the optimal orientation for transgene expression at the ROSA26 locus. ”

“ The Gt (ROSA) 26Sor locus (ROSA26) was first described as a gene trap which is ubiquitously expressed in mouse embryos [15] . As the ROSA26 locus is active in most cells any promoter inserted into the locus should not be restricted in its expression by unfavourable chromatin configurations. This locus has been widely used for expressing endogenous sequences, often reporter genes usually from the endogenous promoter [16] , [17] . The promoter from the ROSA26 locus has been used to drive widespread expression of marker genes in transgenic rats and mice [18] . ”

“ Results Targeting strategy for introducing to ROSA26 locus Establishing dox-dependent gene expression requires two different transgenes, an activator and a responder transgene. Activator constructs were generated with a CMV promoter driving the rtTA2s-M2 variant of the tetracycline transcriptional activator. The CMV promoter was chosen as it works inefficiently in ES cells and should be sensitive to position effects [27] . ”