Construction of a Novel Single Double-Conditional shRNA Expression Vector

Abstract

: Silencing of gene expression by RNA interference RNAi has become a widely used tool for assessing gene function in a fast and easy manner. An important advance in the RNAi eld was the discovery that plasmid-based short hairpin RNA transcription can substitute for synthetic small interference RNAs both in vitro and in vivo. The constitutive knockdown of gene expression by RNAi can limit the scope of experiments, especially if the inhibition of genes leads to cell lethality, which prevents in vivo functional analysis. We have generated double-conditional short hairpin RNA from a single vector that can regulate its own transcription by a combination of tetracycline-inducible and Cre-loxP systems. This vector will be useful for the control of short hairpin RNA expression in a spatially-regulated manner by Cre recombinase as well as a temporally-regulated manner by tetracycline induction. These control features provide for the speci c silencing of genes to facilitate functional genetic analysis in mammals in vivo.

Construction of an All-in-one Double-conditional shRNA Expression Vector

“…The and indicate use of cells that were not induced with IPTG, and those that were induced with IPTG, respectively. adding the NCre expression cassette to the pSingle-tTS-2lox-shRNA vector that we previously constructed 6 . To do so, the site-specific promoter integrated ahead of NCre was replaced between the P sv40 and the Neomycin-resistant region of the original pSingle-tTS-shRNA vector to control the expression of NCre using IPTG in E. coli.…”

“…To overcome these limitations of RNAi, we previously constructed a double-conditional shRNA expression vector 6 from the commercially available pSingle-tTS-shRNA vector Takarabio-Clontech, Inc., Shiga, Japan 7 . This vector consists of two functional units : a tetracycline Tet -induced shRNA expression unit 8 for temporal control, and a recombination unit sequence that is floxed by two loxP-like TATA boxes TATA-lox sequences 9 that can be excised by Cre recombinase 10 for cell-specific control.…”

“…In this study, we attempted to improve on this previously generated double-conditional shRNA expression vector 6 by constructing an all-in-one vector one that contains all essential components within a single vector, by integrating a promoter-specific nuclear localizing Cre NCre expression unit. We confirmed the inducible expression of NCre based on regulation of the lac promoter P lac that can be specifically activated by application of isopropyl--D-thiogalactopyranoside IPTG 11 and also the functional recombination between floxed TATA-lox sequences in Escherichia coli.…”