TISSUE RESTRICTED GENE-EXPRESSION ASSAYED BY DIRECT DNA INJECTION INTO CARDIAC AND SKELETAL-MUSCLE

We have used direct injection of plasmid DNA into heart and tongue in vivo and transfection into cells in culture to determine (1) whether t he pattern of reporter gene expression parallels the cell-type specifi c expression of endogenous genes, (2) whether the pattern of reporter gene expression approximates that of the same constructs transfected i nto myocardiocytes and myogenic cells in culture, (3) whether the expr ession patterns of promoters and enhancers that had been subtly altere d by mutations are similar following transfection and direct DNA injec tion. We utilized reporter gene constructs derived from the two fiber- type specific human troponin C genes: the fast twitch gene (TnCf) and the slow twitch or cardiac gene (cTnC), The endogenous TnCf gene does not express in the heart and the plasmid DNA expresses neither in myoc ardium after injection nor in transfected cardiomyocytes. However inje cted TnCf does express vigorously in tongue muscle. Conversely, the en dogenous cTnC gene does not express in fast twitch skeletal muscles li ke tongue and the plasmid DNA does not express in tongue after injecti on. However, injected cTnC does express in both injected myocardium an d in transfected cardiomyocytes. With few discrepancies, various delet ions and alterations of the promoters and enhancers of these genes tha t have previously been defined by transfection into permissive myogeni c cells in culture gave parallel expression patterns after injection i n myocardium and tongue. Thus we conclude that direct DNA injection ap pears to provide a method to verify the identities of important cis-ac ting regulatory regions that have been mapped in cells in culture.