Intensive RNAi with lentiviral vectors in mammalian cells.
RNAi is a powerful technology for analyzing gene function in human cells. However, its utility can be compromised by inadequate knockdown of the target mRNA or by interpretation of effects without rigorous controls. We review lentiviral vector-based methods that enable transient or stable knockdowns to trace mRNA levels in human CD4+ T cell lines and other targets. Critical controls are reviewed, including rescue of the pre-knockdown phenotype by re-expression of the targeted gene. The time from thinking about a potential knockdown target to analysis of phenotypes can be as short as a few weeks.
Miest
Tanner
T
Department of Molecular Medicine, Mayo Clinic College of Medicine, Guggenheim 1811A, 200 First Street SW, Rochester, MN 55905, USA.
Saenz
Dyana
D
Meehan
Anne
A
Llano
Manuel
M
Poeschla
Eric M
EM
D000818:Animals; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007424:Intracellular Fluid / Q000502:physiology; D016086:Lentivirus / Q000235:genetics*; D008969:Molecular Sequence Data; D034622:RNA Interference / Q000502:physiology*