References of "Bauer, Sebastian"
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See detailOptimization of gene transfer into neonatal rat cardiomyocytes and unmasking of cytomegalovirus promoter silencing.
Bauer, Sebastian; Maier, Sebastian K. G.; Neyses, Ludwig UL et al

in DNA and cell biology (2005), 24(6), 381-7

Cardiomyocytes are notoriously difficult to transfect using standard techniques unless viral vectors such as recombinant adenoviruses are used. Generation of recombinant adenoviruses is, however, a ... [more ▼]

Cardiomyocytes are notoriously difficult to transfect using standard techniques unless viral vectors such as recombinant adenoviruses are used. Generation of recombinant adenoviruses is, however, a complex and time-consuming procedure and not possible for every DNA construct. We therefore optimized DNA/polylysine/adenovirus complexing for efficient gene transfer in neonatal rat cardiomyocytes determining the critical parameters for this method. Importantly, not only the concentration of the various components but also the method used for plasmid purification is critical for this transfection technique. Cesium-chloride-purified DNA is inferior to anion-exchange methods for this purpose possibly because of altered ionic properties. In the second part of this study, we could demonstrate silent gene transfer into cardiomyocytes applying this optimized technique to plasmids encoding luciferase or beta-galactosidase cDNAs under the control of the cytomegalovirus immediate-early promoter. Phorbol myristate acetate and/or forskolin increased the amount of beta-galactosidase positive cells up to fivefold. Luciferase activity could even be increased as much as ninefold. These results demonstrate that the cytomegalovirus promoter is not maximally active in neonatal rat cardiomyocytes under basal conditions. In fact, a large proportion of cells is silently transfected and seems to express (an) inhibitor(s) of transcription from the CMV promoter that can be overcome by stimulation of cAMP- or protein kinase C-dependent pathways. [less ▲]

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See detailRational promoter selection for gene transfer into cardiac cells.
Maass, Alexander; Langer, Stephen J.; Oberdorf-Maass, Silke et al

in Journal of Molecular and Cellular Cardiology (2003), 35(7), 823-31

Cardiomyocytes (CMCs) are extremely difficult to transfect with non-viral techniques, but they are efficiently infected by adenoviruses. The most commonly used promoters to drive protein expression in ... [more ▼]

Cardiomyocytes (CMCs) are extremely difficult to transfect with non-viral techniques, but they are efficiently infected by adenoviruses. The most commonly used promoters to drive protein expression in cardiac myocytes are of viral origin, since they are believed to be constitutively active and minimally regulated by physiological or pharmacological challenge of cells. In recombinant adenoviruses, we systematically compared three different promoters: the cytomegalovirus (CMV), the Rous sarcoma virus (RSV), and a synthetic promoter with three MEF2 transcription factor-binding sites upstream of the heat-shock protein 68 minimal promoter. We determined their basal activity in primary cardiac cells as well as their possible stimulation by commonly used agonists. The CMV promoter was activated up to 60-fold by the phorbol ester phorbol myristate acetate (PMA) and/or forskolin in neonatal rat CMCs and cardiac fibroblasts. Primary adult rat CMCs had higher basal expression from the CMV promoter that was not activated by PMA or forskolin. The RSV promoter was less affected by agonists and was more active in cardiac myocytes compared to cardiac fibroblasts. The MEF2-responsive promoter showed high basal expression in both myocytes and fibroblasts, and minimal induction by phorbol esters and forskolin. The relevance of reporter gene induction was confirmed with a contractile protein, troponin T (TnT). The CMV promoter driving TnT could be induced more than 15-fold with phenylephrine or forskolin to replace the endogenous protein almost to completion at a multiplicity of infection of 10. These results suggest the following use of the tested promoters: an inducible system (CMV), a myocyte-enriched system (RSV), or a stable control system (MEF2). [less ▲]

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