Gene silencing by RNA interference RNAi is widely used for assessing gene function. An important advance in the RNAi eld was the discovery that plasmid-based RNAi can substitute for synthetic small interfering RNA in vitro and in vivo. However, constitutive and ubiquitous knockdown of gene expression by RNAi in mice can limit the scope of experiments because this process can lead to embryonic lethality, or result in compensatory overexpression of other genes such that no phenotypic

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... occur. Either way, analyses of the physiological roles of the gene of interest in adult mice are not possible. To overcome these limitations, we previously constructed a double-conditional short-hairpin RNA shRNA expression vector that can regulate shRNA expression in a spatiotemporal manner with a tetracycline-inducible floxed stuffer sequence selectively excised by application of Cre recombinase. In this study, we aimed to modify this vector to create an all-in-one vector that produces double-conditional transgenic mice through a single round of gene transfer to fertilized eggs. We added a coding region for nuclear localizing Cre NCre recombinase with a multi-cloning site for a cell-specific promoter into the double-conditional short-hairpin RNA shRNA expression vector that we previously constructed. Using Escherichia coli, we con rmed successful construction of the vector. First, we con rmed isopropyl--D-thiogalactopyranoside-induced expression of NCre recombinase through the lac operon as a specific promoter by western blotting. Second, we confirmed functional recombination of the oxed sequence of loxP-like TATA-lox by analysing restriction enzyme-digested fragments. This all-in-one double-conditional shRNA expression vector will be useful for reversible in vitro and in vivo knockdown of target gene expression, in target cells via promoter-speci c expression of NCre, and at speci c times by tetracycline application.