Lentivirus production.
RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.
Diabetes Center, University of California, San Francisco, USA.
Xiaoyin
X
Wang
Michael
M
McManus
D002460:Cell Line; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection / Q000379:methods; D014773:Virology / Q000379:methods*
D034741:RNA, Small Interfering