| Chapter title |
Silencing Genes in the Heart.
|
|---|---|
| Chapter number | 2 |
| Book title |
Cardiac Gene Therapy
|
| Published in |
Methods in molecular biology, January 2017
|
| DOI | 10.1007/978-1-4939-6588-5_2 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-6586-1, 978-1-4939-6588-5
|
| Authors |
Henry Fechner D.V.M., Roland Vetter, Jens Kurreck, Wolfgang Poller, Henry Fechner |
| Editors |
Kiyotake Ishikawa |
| 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. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 7 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Professor > Associate Professor | 3 | 43% |
| Researcher | 1 | 14% |
| Student > Doctoral Student | 1 | 14% |
| Unknown | 2 | 29% |
| Readers by discipline | Count | As % |
|---|---|---|
| Medicine and Dentistry | 2 | 29% |
| Neuroscience | 1 | 14% |
| Biochemistry, Genetics and Molecular Biology | 1 | 14% |
| Unknown | 3 | 43% |