Figure 1.
Vector configuration, ROSA26 exchange and recombination events.
(A) Exchange constructs harbouring the promoter-luciferase cassettes in either sense or antisense orientation were targeted to the ROSA26 locus via PhiC31 integrase mediated cassette exchange. (B) A Fluc coding sequence was targeted to the ROSA26 locus via PhiC31 integrase mediated cassette exchange. Flp recombinase mediated deletion of the selection cassette, brought the luciferase cassette into the context of the ROSA26 forward transcript via the 5′ Splice Acceptor (SA).
Figure 2.
Relative promoter activities of Fluc reporter constructs stably integrated at ROSA26.
Fluc expression cassettes under the control of different promoters were inserted into the ROSA26 locus in both sense and antisense orientation by PhiC31 integrase mediated cassette exchange. Fluc activity from the exogenous promoters is expressed relative to that of luciferase under the control of the endogenous ROSA26 promoter (ROSA26>Fluc). “Fluc Only” represents ES cells harbouring the promoterless construct, ROSA26>Neo>Fluc represents ES cells harbouring inactive ROSA26 driven Fluc, prior to Flp deletion, and IDG26.10-3 ES cells are the “wild-type” ES cells used prior to the transgene insertion. Using multilevel modelling, the difference in activities between promoter-luciferase cassettes positioned in the sense and antisense orientations were found to be highly significant for EF1α (***p = 0.0007) and significant for UbC and MC1(*). Fluc assays were normalized to total protein concentrations and error bars represent the standard error of the mean from at least 3 independent experiments.
Figure 3.
Relative promoter activities of transiently transfected Fluc reporter constructs.
Fluc expression cassettes under the control of different promoters were transiently transfected into IDG26.10-3 ES cells, co-transfected with an Rluc expression plasmid to control for transfection efficiency and cell number. All Fluc readings were normalized against Rluc readings and promoter activities were expressed relative to the activity from the PGK promoter in the sense orientation for comparison. pCB9-CB-Fluc is the construct used to generate the cell line which expresses Fluc under the control of the endogenous ROSA26 promoter. pCB92 is the empty exchange vector without a promoter or Fluc coding sequence. Error bars represent the standard error of the mean from at least 3 independent experiments.
Table 1.
Relative strengths of the different promoters in mouse ES cells, either stably integrated into ROSA26 or transiently transfected.