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 the pattern of reporter gene expression parallels the cell-type specific expression of endogenous genes, (2) whether the pattern of reporter gene expression approximates that of the same constructs transfected into myocardiocytes and myogenic cells in culture, (3) whether the expression patterns of promoters and enhancers that had been subtly altered by mutations are similar following transfection and direct DNA injection. 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 myocardium after injection nor in transfected cardiomyocytes. However injected TnCf does express vigorously in tongue muscle. Conversely, the endogenous cTnC gene does not express in fast twitch skeletal muscles like tongue and the plasmid DNA does not express in tongue after injection. However, injected cTnC does express in both injected myocardium and in transfected cardiomyocytes. With few discrepancies, various deletions and alterations of the promoters and enhancers of these genes that have previously been defined by transfection into permissive myogenic cells in culture gave parallel expression patterns after injection in myocardium and tongue. Thus we conclude that direct DNA injection appears to provide a method to verify the identities of important cis-acting regulatory regions that have been mapped in cells in culture.
Prentice
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H
Institute for Genetic Medicine, University of Southern California School of Medicine, Los Angeles 90033.
Kloner
R A
RA
Prigozy
T
T
Christensen
T
T
Newman
L
L
Li
Y
Y
Kedes
L
L
D000818:Animals; D004274:DNA, Recombinant / Q000008:administration & dosage / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D008845:Microinjections; D009132:Muscles / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D009928:Organ Specificity; D010957:Plasmids; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014059:Tongue / Q000378:metabolism; D014336:Troponin / Q000096:biosynthesis* / Q000145:classification / Q000235:genetics; D019209:Troponin C
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D014336:Troponin; D019209:Troponin C