DIRECT DNA INJECTION INTO MOUSE TONGUE MUSCLE FOR ANALYSIS OF PROMOTER FUNCTION INVIVO

The striated muscle of the tongue provides a readily accessible site f or the introduction of DNA expression vectors. Parameters were establi shed to use the striated muscle of the tongue as a model system for th e examination of gene expression following the direct injection of DNA constructs bearing gene promoter sequences controlling the expression of reporter genes. Plasmid expression vectors were used that containe d either constitutive or muscle-specific promoters directing the trans cription of reporter genes. Chloramphenicol acetyltransferase (CAT), l uciferase, and beta-galactosidase (lacZ) were used as the reporter gen es to detect the promoter-specific expression of the injected DNA. The expression of the injected plasmids was directly correlated with the mass of injected DNA and the time of incubation following the injectio n. Maximal levels of reporter gene expression were observed seven days after the injection, and the expression was maintained for more than two months following injection. Simultaneous injection of two individu al expression vectors bearing either CAT or luciferase reporter genes resulted in a dose-dependent level of expression for each of the plasm ids. The linearity of the coexpression provided a means to normalize D NA uptake and analyze promoter efficiency. The troponin C-fast enhance r linked to its own promoter directed significantly more CAT expressio n than an enhancerless SV40 promoter-CAT plasmid, demonstrating that d ifferent promoter strengths could be determined in the mouse tongue mu scle in vivo. This model system represents a convenient means to appro ach the functional analysis of muscle gene promoters in vivo.