An important advance in the RNA interference (RNAi) field was the discovery that plasmid vector-mediated expression of short hairpin RNA (shRNA) can substitute for synthetic small interference RNA (siRNA) s in vitro and in vivo. But the constitutive and/or ubiquitous knockdown of target gene by this method has still limited the utility, especially if the inhibition of target gene leads to lethality, which prevents in vivo functional analysis. To overcome these limitations, the time (or period)- and cell (or tissue)- specific regulation of shRNA expression should have been required as the double-conditional mice. Conventional protocols for such an artificial regulation of gene expression in vivo are based on the tetracycline-controlled reversible system for the time- and the Cre-loxP system for cell-specificities respectively. The double-conditional transgenic mice are expected by mating their two lines of genetic engineered mice at last. Therefore, we have designed the "All-in-One" type single double-conditional shRNA expression vectors having both systems within a single vector format to make the transgenic mice with double-conditional RNAi without much effort.