Lentivirus-delivered stable gene silencing by RNAi in primary cells.
Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.
Stewart
Sheila A
SA
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA. sstewart@wi.mit.edu
Dykxhoorn
Derek M
DM
Palliser
Deborah
D
Mizuno
Hana
H
Yu
Evan Y
EY
An
Dong Sung
DS
Sabatini
David M
DM
Chen
Irvin S Y
IS
Hahn
William C
WC
Sharp
Phillip A
PA
Weinberg
Robert A
RA
Novina
Carl D
CD
D003713:Dendritic Cells; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D016086:Lentivirus / Q000235:genetics* / Q000502:physiology*; D034622:RNA Interference / Q000502:physiology*