Chang, K., Marran, K., Valentine, A., Hannon, G. J. (2013) Packaging shRNA retroviruses. Cold Spring Harbor Protocols, 2013 (8). pp. 734-737. ISSN 19403402
Abstract
To silence a mammalian gene by RNAi using an encoded trigger, a short-hairpin RNA (shRNA) is integrated into the host cell genome as a stable transgene. Target cells are infected with viral plasmid containing shRNA inserted into the vector backbone. Before infection, the plasmid is transfected into a packaging cell line, which provides the trans-acting factors necessary for virus production. These include, minimally, capsid proteins and reverse transcriptase, but they can also include other regulatory factors (e.g., tat for some lentiviral vectors). It is critical to choose the correct packaging cell system for the viral backbone to be used. The packaging cell also defines the host range of the virus, depending on the envelope protein that it expresses. Ecotropic viruses are limited to rodent hosts, whereas amphotropic viruses have a broader host range that also includes humans. Often, investigators will express a nonretroviral envelope, such as vesicular stomatitus virus (VSV) glycoprotein, to enhance virus stability and host range and to enable viruses to be concentrated following production. Although viruses carrying shRNAs are packaged almost identically to viruses carrying protein-encoding genes, one twist is worth noting. shRNAs are efficiently cleaved by the host RNAi biogenesis machinery, which can reduce the level of viral genomic RNAs and consequently viral titers. Therefore, titers can be enhanced by cotransfecting the viral plasmid with a small interfering RNA (siRNA) that targets DGCR- 8/Pasha, which is a core microRNA (miRNA) biogenesis component. siRNAs against Drosha can also be used.
| Item Type: | Paper |
|---|---|
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNA regulation organism description > virus > retrovirus |
| CSHL Authors: | |
| Communities: | CSHL labs > Chang lab CSHL labs > Hannon lab |
| Depositing User: | Adrian Gomez |
| Date: | 2013 |
| Date Deposited: | 22 Nov 2019 20:19 |
| Last Modified: | 22 Nov 2019 20:19 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/38725 |
Actions (login required)
 |
Administrator's edit/view item |