ENHANCER AND PROMOTER CHIMERAS IN PLASMIDS DESIGNED FOR INTRAMUSCULARINJECTION - A COMPARATIVE IN-VIVO AND IN-VITRO STUDY

Enhancers and promoters from various muscle-specific genes were substi tuted for or combined with the enhancer/promoter of the human cytomega lovirus (CMV) IE gene in a luciferase reporter gene plasmid in an effo rt to identify new promoter chimeras with increased expression activit y after direct intramuscular injection. The regulatory sequence substi tutions or additions varied in content, location, and orientation rela tive to the CMV regulatory sequences. The expression activities of the derivative and parent plasmids were compared quantitatively in vivo u sing a standard mouse intramuscular injection assay, and lit vitro by transfection of differentiated C2C12 mouse myoblasts and BHK hamster K idney cells, to test whether cultured cell transfection could substitu te for at least some animal experimentation. In vivo, 1 of 19 of the e nhancer/promoter chimeras increased expression levels, In vitro, some chimeras showed significant expression augmentation in C2C12 cells, bu t not in BHK cells, We conclude that because of differences in plasmid expression profiles, these cell culture systems cannot readily substi tute for irt vivo testing of new plasmid constructs.