title: Silencing Genes in the Heart.
abstract: Silencing of cardiac genes by RNA interference (RNAi) has developed into a powerful new method to treat cardiac diseases. Small interfering (si)RNAs are the inducers of RNAi, but cultured primary cardiomyocytes and heart are highly resistant to siRNA transfection. This can be overcome by delivery of small hairpin (sh)RNAs or artificial microRNA (amiRNAs) by cardiotropic adeno-associated virus (AAV) vectors. Here we describe as example of the silencing of a cardiac gene, the generation and cloning of shRNA, and amiRNAs directed against the cardiac protein phospholamban. We further describe the generation of AAV shuttle plasmids with self complementary vector genomes, the production of AAV vectors in roller bottles, and their purification via iodixanol gradient centrifugation and concentration with filter systems. Finally we describe the preparation of primary neonatal rat cardiomyocytes (PNRC), the transduction of PNRC with AAV vectors, and the maintenance of the transduced cell culture.
authors.affiliation: Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355, Berlin, Germany. henry.fechner@tu-berlin.de.
authors.forename: Henry
authors.initials: H
authors.lastname: Fechner
authors.affiliation: Institute of Clinical Pharmacology & Toxicology, Charité - Universitätsmedizin Berlin, Campus Charité Mitte, Charitéplatz 1, 10117, Berlin, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Vetter
authors.affiliation: Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355, Berlin, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Kurreck
authors.affiliation: Department of Cardiology & Pneumology, Charité - Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200, Berlin, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Poller
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D002469:Cell Separation; D002478:Cells, Cultured; D011232:Chemical Precipitation; D003001:Cloning, Molecular; D000229:Dependovirus / Q000235:genetics; D005374:Filtration; D020868:Gene Silencing; D057809:HEK293 Cells; D006321:Heart / Q000502:physiology*; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D032383:Myocytes, Cardiac / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D014161:Transduction, Genetic
chemical_list: D035683:MicroRNAs; D034741:RNA, Small Interfering
keywords: AAV vectors; Iodixanol gradient centrifugation; Primary neonatal rat cardiomyocytes; amiRNA; shRNA; siRNA