Control of siRNA expression using the Cre–loxP recombination system

V Kasim, M Miyagishi, K Taira - Nucleic acids research, 2004 - academic.oup.com
Nucleic acids research, 2004academic.oup.com
Gene silencing mediated by RNA interference (RNAi) was first discovered in Caenorhabditis
elegans, and was subsequently recognized in various other organisms. In mammalian cells,
RNAi can be induced by small interfering RNAs (siRNAs). In earlier studies, our group
developed a vector‐based system for expression of siRNA under control of a polymerase III
promoter, the U6 promoter, which can induce RNAi in living cells. We here describe a
system for controlling the U6 promoter‐driven expression of siRNA using the Cre–loxP …
Abstract
Gene silencing mediated by RNA interference (RNAi) was first discovered in Caenorhabditis elegans, and was subsequently recognized in various other organisms. In mammalian cells, RNAi can be induced by small interfering RNAs (siRNAs). In earlier studies, our group developed a vector‐based system for expression of siRNA under control of a polymerase III promoter, the U6 promoter, which can induce RNAi in living cells. We here describe a system for controlling the U6 promoter‐driven expression of siRNA using the Cre–loxP recombination system. We constructed a ‘Cre‐On’ siRNA expression vector which could be switched on upon excision catalyzed by Cre recombinase, which was delivered to cells directly from the medium as a fusion protein. An examination of the effectiveness of RNAi against a reporter gene revealed that addition of TAT‐NLS‐Cre (where NLS is a nuclear localization signal and TAT is a peptide of 11 amino acids derived from HIV) to the medium resulted in plasmid recombination, generation of siRNA and suppression of reporter activity. This system should allow us to induce RNAi in a spatially, temporally, cell type‐specifically or tissue‐specifically controlled manner and potentiate the improved application of RNAi in both an experimental and a therapeutic context.
Oxford University Press