UNIT 5.26 Lentiviral Strategies for RNAi Knockdown of Neuronal Genes
Published Online: 1 APR 2007
Copyright © 2007 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Neuroscience
How to Cite
Crittenden, J. R., Heidersbach, A. and McManus, M. T. 2007. Lentiviral Strategies for RNAi Knockdown of Neuronal Genes. Current Protocols in Neuroscience. 39:5.26:5.26.1–5.26.21.
- Published Online: 1 APR 2007
- Published Print: APR 2007
RNA interference (RNAi) refers to the process by which 21- to 23-nucleotide short interfering RNAs (siRNAs) mediate post-transcriptional degradation of homologous mRNA transcripts. This process is carried out by an endogenous pathway that centers on the use of endogenously encoded small RNAs, and can be hijacked to knock down the expression of any target protein by introducing a specific siRNA into a cell. Stable knockdown can be obtained by constitutive expression of the siRNA from the host chromosome. Retroviruses, such as lentivirus, provide a convenient vector by which to integrate RNAi expression constructs. Lentiviruses can infect nondividing cells, thereby allowing knockdown in cells such as mature neurons. This unit provides methods to design and clone siRNAs into a lentiviral vector. Additional protocols describe production and titering of the lentivirus, as well as safety testing. Finally, methods are provided for infecting neurons in culture and in vivo with RNAi lentivirus.