Figure 6. OriLyt dependent transgene expression in vivo.

Generation of virus inducible oriLyt dependent luciferase animal (VIOLA). (A) (1.) mES cell clones were transfected with pEpibo-luc-ori and (2.) pre-selected for their induction capacity by MCMV infection in vitro. (3.) Mouse lines were analyzed for expression of FL before and after infection in explant cultures of different organs. (B) mES cells were transfected with pEpibo-luc-ori and 8 clones were isolated. A portion of the clones were partially differentiated for 3 weeks and then infected with MCMV at an MOI of 0.5 or left untreated. 36 h p.i. a bioluminescence assay was performed and the induction of the FL was calculated as the ratio of RLU of infected to uninfected cells. mES clones A3 and B8 were selected for generation of transgenic animals. (C) Explant cultures of lungs taken from mice of the Viola–A strain, backcrossed one (2^nd generation) or two (3^rd generation) times to 129X1/SvJ, were produced and were infected with MCMV-wt with an MOI of 0.5 (hatched bars) or left untreated (white bars). 24 h p.i. FL expression was determined by measuring relative light units (RLU). As a control mice of the background strain 129X1/SvJ (wt/wt) were used. (D) Explant cultures from bone marrow (BM), heart (He), muscle (Mu), fat (FA), spleen (Sp) and salivary glands (Sg) were grown from a VIOLA-A mouse of the fourth generation. Cells were infected with MCMV-wt with MOI of 0.5 (hatched bars) or left untreated (white bars). 24 h p.i. FL expression was determined by measuring relative light units (RLU). (p.i. = post infection ; BG = background of luciferin).