© 1996 American Heart Association, Inc.
Articles |
Novel Adenovirus Component System That Transfects Cultured Cardiac Cells With High Efficiency
From the Department of Biochemistry and Molecular Biology (T.A.K., S.T.G., T.B.R.) and the Department of Physiology (T.A.K., W.J.L.), University of Maryland School of Medicine, Baltimore, and Dupont/Merck (J.J.O.), Research and Development, Glenholden Laboratory, Glenholden, Pa.
Correspondence to Dr Terry B. Rogers, Department of Biological Chemistry, University of Maryland School of Medicine, 108 N Greene St, Baltimore, MD 21201. E-mail trogers@umabnet.ab.umd.edu.
Abstract 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 ß-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-ß-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
. 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.
Key Words: transfection • adenovirus • myocytes • gene expression
This article has been cited by other articles:
 |
|
 |
|
  N. D. Glaser, Y. O. Lukyanenko, Y. Wang, G. M. Wilson, and T. B. Rogers JNK activation decreases PP2A regulatory subunit B56{alpha} expression and mRNA stability and increases AUF1 expression in cardiomyocytes Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1183 - H1192. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Gigena, A. Ito, H. Nojima, and T. B. Rogers A B56 regulatory subunit of protein phosphatase 2A localizes to nuclear speckles in cardiomyocytes Am J Physiol Heart Circ Physiol, July 1, 2005; 289(1): H285 - H294. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Li, D. Georgakopoulos, G. Lu, L. Hester, D. A. Kass, J. Hasday, and Y. Wang p38 MAP Kinase Mediates Inflammatory Cytokine Induction in Cardiomyocytes and Extracellular Matrix Remodeling in Heart Circulation, May 17, 2005; 111(19): 2494 - 2502. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. G. Petrich, X. Gong, D. L. Lerner, X. Wang, J. H. Brown, J. E. Saffitz, and Y. Wang c-Jun N-Terminal Kinase Activation Mediates Downregulation of Connexin43 in Cardiomyocytes Circ. Res., October 4, 2002; 91(7): 640 - 647. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. A. Kohout, F.-T. Lin, S. J. Perry, D. A. Conner, and R. J. Lefkowitz beta -Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking PNAS, February 1, 2001; (2001) 41608198. [Abstract] [Full Text]
|
|
|
|
 |
|
 D. JAFFUEL, P. DEMOLY, C. GOUGAT, P. BALAGUER, G. MAUTINO, P. GODARD, J. BOUSQUET, and M. MATHIEU Transcriptional Potencies of Inhaled Glucocorticoids Am. J. Respir. Crit. Care Med., July 1, 2000; 162(1): 57 - 63. [Abstract] [Full Text]
|
|
|
|
|
|
Y.-Y. Zhou, S.-Q. Wang, W.-Z. Zhu, A. Chruscinski, B. K. Kobilka, B. Ziman, S. Wang, E. G. Lakatta, H. Cheng, and R.-P. Xiao Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology Am J Physiol Heart Circ Physiol, July 1, 2000; 279(1): H429 - H436. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-k. Wei, H. M. Colecraft, C. D. DeMaria, B. Z. Peterson, R. Zhang, T. A. Kohout, T. B. Rogers, and D. T. Yue Ca2+ Channel Modulation by Recombinant Auxiliary {beta} Subunits Expressed in Young Adult Heart Cells Circ. Res., February 4, 2000; 86(2): 175 - 184. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. D. Luo, Y. Wang, G. Werlen, S. Camp, K. R. Chien, and P. Taylor Calcineurin Enhances Acetylcholinesterase mRNA Stability during C2-C12 Muscle Cell Differentiation Mol. Pharmacol., November 1, 1999; 56(5): 886 - 894. [Abstract] [Full Text]
|
|
|
|
|
|
D. Jaffuel, P. Demoly, C. Gougat, G. Mautino, J. Bousquet, and M. Mathieu Rifampicin Is Not an Activator of the Glucocorticoid Receptor in A549 Human Alveolar Cells Mol. Pharmacol., May 1, 1999; 55(5): 841 - 846. [Abstract] [Full Text]
|
|
|
|
|
|
K. Kunisada, E. Tone, Y. Fujio, H. Matsui, K. Yamauchi-Takihara, and T. Kishimoto Activation of gp130 Transduces Hypertrophic Signals via STAT3 in Cardiac Myocytes Circulation, July 28, 1998; 98(4): 346 - 352. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Eble, M. Qi, S. Waldschmidt, P. A. Lucchesi, K. L. Byron, and A. M. Samarel Contractile activity is required for sarcomeric assembly in phenylephrine-induced cardiac myocyte hypertrophy Am J Physiol Cell Physiol, May 1, 1998; 274(5): C1226 - C1237. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Inesi, D. Lewis, C. Sumbilla, A. Nandi, C. Strock, K. W. Huff, T. B. Rogers, D. C. Johns, P. D. Kessler, and C. P. Ordahl Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes Am J Physiol Cell Physiol, March 1, 1998; 274(3): C645 - C653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Fasbender, J. Zabner, M. Chillon, T. O. Moninger, A. P. Puga, B. L. Davidson, and M. J. Welsh Complexes of Adenovirus with Polycationic Polymers and Cationic Lipids Increase the Efficiency of Gene Transfer in Vitro and in Vivo J. Biol. Chem., March 7, 1997; 272(10): 6479 - 6489. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. O. Rybin, X. Xu, M. P. Lisanti, and S. F. Steinberg Differential Targeting of beta -Adrenergic Receptor Subtypes and Adenylyl Cyclase to Cardiomyocyte Caveolae. A MECHANISM TO FUNCTIONALLY REGULATE THE cAMP SIGNALING PATHWAY J. Biol. Chem., December 22, 2000; 275(52): 41447 - 41457. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. de Jager, T. Pelzer, S. Muller-Botz, A. Imam, J. Muck, and L. Neyses Mechanisms of Estrogen Receptor Action in the Myocardium. RAPID GENE ACTIVATION VIA THE ERK1/2 PATHWAY AND SERUM RESPONSE ELEMENTS J. Biol. Chem., July 20, 2001; 276(30): 27873 - 27880. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Kohout, F.-T. Lin, S. J. Perry, D. A. Conner, and R. J. Lefkowitz beta -Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking PNAS, February 13, 2001; 98(4): 1601 - 1606. [Abstract] [Full Text] [PDF]
|
|