Stanislas Werfel1, Andreas Jungmann2, Lorenz Lehmann3, Jan Ksienzyk2, Raffi Bekeredjian3, Ziya Kaya3, Barbara Leuchs4, Alfred Nordheim5, Johannes Backs3, Stefan Engelhardt6, Hugo A Katus3, Oliver J Müller7. 1. Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg 69120, Germany Institute for Pharmacology and Toxicology, Technische Universität München, Biedersteiner Str. 29, Munich 80802, Germany DZHK (German Centre for Cardiovascular Research), Partner Site Munich Heart Alliance, Munich, Germany. 2. Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg 69120, Germany. 3. Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg 69120, Germany DZHK (German Center for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Germany. 4. Applied Tumorvirology, German Cancer Research Center, Heidelberg, Germany. 5. Interfaculty Institute for Cell Biology, Department of Molecular Biology, University of Tübingen, Tübingen, Germany. 6. Institute for Pharmacology and Toxicology, Technische Universität München, Biedersteiner Str. 29, Munich 80802, Germany DZHK (German Centre for Cardiovascular Research), Partner Site Munich Heart Alliance, Munich, Germany. 7. Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg 69120, Germany DZHK (German Center for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Germany oliver.mueller@med.uni-heidelberg.de o.mueller@dkfz-heidelberg.de.
Abstract
AIMS: Inducible gene targeting in mice using the Cre/LoxP system has become a valuable tool to analyse the roles of specific genes in the adult heart. However, the commonly used Myh6-MerCreMer system requires time-consuming breeding schedules and is potentially associated with cardiac side effects, which may result in transient cardiac dysfunction. The aim of our study was to establish a rapid and simple system for cardiac gene inactivation in conditional knockout mice by gene transfer of a Cre recombinase gene using adeno-associated viral vectors of serotype 9 (AAV9). METHODS AND RESULTS: AAV9 vectors expressing Cre under the control of a human cardiac troponin T promoter (AAV-TnT-Cre) enabled a highly efficient Cre/LoxP switching in cardiomyocytes 2 weeks after injection into 5- to 6-week-old ROSA26-LacZ reporter mice. Recombination efficiency was at least as high as observed with the Myh6-MerCreMer system. No adverse side effects were detected upon application of AAV-TnT-Cre. As proof of principle, we studied AAV-TnT-Cre in a conditional knockout model (Srf-flex1 mice) to deplete the myocardium of the transcription factor serum response factor (SRF). Four weeks after AAV-TnT-Cre injection, a strong decrease in the cardiac expression of SRF mRNA and protein was observed. Furthermore, mice developed a severe cardiac dysfunction with increased interstitial fibrosis in accordance with the central role of SRF for the expression of contractile and calcium trafficking proteins in the heart. CONCLUSIONS: AAV9-mediated expression of Cre is a promising approach for rapid and efficient conditional cardiac gene knockout in adult mice. Published on behalf of the European Society of Cardiology. All rights reserved.
AIMS: Inducible gene targeting in mice using the Cre/LoxP system has become a valuable tool to analyse the roles of specific genes in the adult heart. However, the commonly used Myh6-MerCreMer system requires time-consuming breeding schedules and is potentially associated with cardiac side effects, which may result in transient cardiac dysfunction. The aim of our study was to establish a rapid and simple system for cardiac gene inactivation in conditional knockout mice by gene transfer of a Cre recombinase gene using adeno-associated viral vectors of serotype 9 (AAV9). METHODS AND RESULTS: AAV9 vectors expressing Cre under the control of a human cardiac troponin T promoter (AAV-TnT-Cre) enabled a highly efficient Cre/LoxP switching in cardiomyocytes 2 weeks after injection into 5- to 6-week-old ROSA26-LacZ reporter mice. Recombination efficiency was at least as high as observed with the Myh6-MerCreMer system. No adverse side effects were detected upon application of AAV-TnT-Cre. As proof of principle, we studied AAV-TnT-Cre in a conditional knockout model (Srf-flex1 mice) to deplete the myocardium of the transcription factor serum response factor (SRF). Four weeks after AAV-TnT-Cre injection, a strong decrease in the cardiac expression of SRF mRNA and protein was observed. Furthermore, mice developed a severe cardiac dysfunction with increased interstitial fibrosis in accordance with the central role of SRF for the expression of contractile and calcium trafficking proteins in the heart. CONCLUSIONS: AAV9-mediated expression of Cre is a promising approach for rapid and efficient conditional cardiac gene knockout in adult mice. Published on behalf of the European Society of Cardiology. All rights reserved.
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