Design and cloning of lentiviral vectors expressing small interfering RNAs.
RNA interference (RNAi) has emerged as a powerful technique to downregulate gene expression. The use of polIII promoters to express small hairpin RNAs (shRNAs), combined with the versatility and robustness of lentiviral vector-mediated gene delivery to a wide range of cell types offers the possibility of long-term downregulation of specific target genes both in vitro and in vivo. The use of silencing lentivectors allows for a rapid and convenient way of establishing cell lines (or transgenic mice) that stably express shRNAs for analysis of phenotypes produced by knockdown of a gene product. Here we present two possible protocols describing the design and cloning of silencing lentiviral vectors. These protocols can be completed in less than 3 weeks.
Tiscornia
Gustavo
G
The Salk Institute for Biological Studies, Laboratory of Genetics, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.
Singer
Oded
O
Verma
Inder M
IM
D003001:Cloning, Molecular / Q000379:methods*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016086:Lentivirus / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism
D034741:RNA, Small Interfering