Novel adenovirus component system that transfects cultured cardiac cells with high efficiency.
Although it is clear that gene transfection is a potentially valuable approach in the study of cardiac cell function and differentiation, classic transfection methods are limited by their poor efficiencies in cardiac cells. Recent studies show that recombinant replication-defective human adenovirus can transfect primary cardiac cultures with near 100% efficiency. Since such recombinants are time consuming to prepare, the goal of this study was to develop a plasmid/viral transfection system that would capitalize on the advantages of adenovirus. We have found that a "component system" formed by preincubation of Ad5dl312 adenovirus, poly-L-lysine, and an expression plasmid (lacZ reporter gene under control of the human cytomegalovirus (HCMV) major immediate early promoter) can transfect cultured cardiac cells. Optimal conditions were determined by quantifying beta-galactosidase expression. Histochemical analysis of cultures revealed that the component system transfected 70% of the cells under these conditions. LacZ-positive myocytes could be identified in intact myocytes with the fluorescent substrate C12-fluorescein di-beta-galactopyranoside. Functional studies with such cells indicated that contractile behavior was maintained in transfected cardiocytes. Furthermore, the component system was used to transfect a DNA vector expressing a physiologically relevant protein, protein kinase C delta. In summary, this powerful and simple approach can promote the expression of heterologous genes that can be studied at the biochemical and cellular level in cardiac cells.
Kohout
T A
TA
Department of Biochemistry, University of Maryland School of Medicine, Baltimore 21201, USA. trogers@umabnet.ab.umd.edu
O'Brian
J J
JJ
Gaa
S T
ST
Lederer
W J
WJ
Rogers
T B
TB
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D002478:Cells, Cultured; D006801:Humans; D009206:Myocardium / Q000378:metabolism*; D010957:Plasmids; D011493:Protein Kinase C; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D011493:Protein Kinase C; D001616:beta-Galactosidase