A loxP-containing pol II promoter for RNA interference is reversibly regulated by Cre recombinase.
Several DNA vectors for RNA interference in mammalian cells have been described. These express a short hairpin RNA (shRNA) that is subsequently processed into mature small interfering RNAs (siRNAs). We previously developed the siRNA-expressing vector psiUx based on the polII promoter of the U1 small nuclear RNA gene. Here we describe the conversion of such construct into an inducible system. The starting construct psiUStuff contains a loxP-Stuffer-loxP cassette just upstream the transcription initiation site and does not express the shRNA until the two canonical loxP sites undergo Cre-mediated recombination. If sustained expression of the recombinase is maintained, transcription is repressed and shRNA synthesis is abolished. Therefore, in our system the Cre recombinase exhibits the dual function of activator and repressor allowing the on/off regulation of siRNAs production. Using a Cre recombinase whose transcription is under the control of a tetOn system, we show the temporally controlled expression of an shRNA directed towards the lamin A/C mRNA, as well as the regulated knockdown of its target.
Iovino
Nicola
N
I.R.B.M. P. Angeletti, Pomezia, Italy. nicola.iovino@rockefeller.edu
Denti
Michela Alessandra
MA
Bozzoni
Irene
I
Cortese
Riccardo
R
D004257:DNA Polymerase II / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D019426:Integrases / Q000502:physiology*; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D011995:Recombination, Genetic
C045073:Cre recombinase; D019426:Integrases; D004257:DNA Polymerase II