title: Rapid and highly efficient inducible cardiac gene knockout in adult mice using AAV-mediated expression of Cre recombinase.
abstract: OBJECTIVE
  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).
  
  
  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.
authors.affiliation: 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.
authors.forename: Stanislas
authors.initials: S
authors.lastname: Werfel
authors.affiliation: Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg 69120, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Jungmann
authors.affiliation: 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.
authors.forename: Lorenz
authors.initials: L
authors.lastname: Lehmann
authors.affiliation: Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg 69120, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Ksienzyk
authors.affiliation: 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.
authors.forename: Raffi
authors.initials: R
authors.lastname: Bekeredjian
authors.affiliation: 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.
authors.forename: Ziya
authors.initials: Z
authors.lastname: Kaya
authors.affiliation: Applied Tumorvirology, German Cancer Research Center, Heidelberg, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Leuchs
authors.affiliation: Interfaculty Institute for Cell Biology, Department of Molecular Biology, University of Tübingen, Tübingen, Germany.
authors.forename: Alfred
authors.initials: A
authors.lastname: Nordheim
authors.affiliation: 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.
authors.forename: Johannes
authors.initials: J
authors.lastname: Backs
authors.affiliation: 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.
authors.forename: Stefan
authors.initials: S
authors.lastname: Engelhardt
authors.affiliation: 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.
authors.forename: Hugo A
authors.initials: HA
authors.lastname: Katus
authors.affiliation: 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.
authors.forename: Oliver J
authors.initials: OJ
authors.lastname: Müller
mesh_terms: D000818:Animals; D009202:Cardiomyopathies / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D000229:Dependovirus / Q000201:enzymology / Q000235:genetics*; D015536:Down-Regulation; D005355:Fibrosis; D055785:Gene Knockdown Techniques; D005838:Genotype; D019426:Integrases / Q000096:biosynthesis / Q000235:genetics*; D007763:Lac Operon; D008297:Male; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology; D010641:Phenotype; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D022661:RNA, Untranslated / Q000235:genetics; D026362:Serum Response Factor / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D020107:Troponin T / Q000235:genetics; D018487:Ventricular Dysfunction, Left / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D016277:Ventricular Function, Left
chemical_list: C110853:Gt(ROSA)26Sor non-coding RNA, mouse; D012333:RNA, Messenger; D022661:RNA, Untranslated; D026362:Serum Response Factor; D020107:Troponin T; C045073:Cre recombinase; D019426:Integrases
keywords: Adeno-associated virus; Cardiomyopathy; Conditional transgenic mouse; Gene regulation; Serum response factor