Design and constructs of the single “All-in-One” type double conditional shRNA expression vectors for a spatio-temporal gene and/or cell targeting

抄録

The functional identification of individual genes and/or cells in multicellular organism will give us invaluable pharmacological information for translational research into drug target discovery. Silencing of specific gene expression by RNA interference (RNAi) has now become a widely used tool for assessing the gene function in a fast and easy manner. An important advance in the 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 knock-down of target gene by this method have 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 (Tet)-controlled reversible system for the time- and the Cre-loxP system for cell-specificities respectively, and 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. In these vectors, we have also tried to avoid the potential toxic effects of Cre not only by the use of self-deleting Cre, which deletes itself from the host cell after it is expressed, but also by the modifications of Cre recombinase as Cre-MODC, DD-Cre or Cre-ERT2. We have also applied the Piggyback (PB)-transposon system for integrating multiple functional units contained the core of these single double conditional shRNA expression vectors into genomes with the use of PB transposase mRNA which was short-lived without integration of PB transposase gene itself. Such the low toxic and highly integrated systems may be useful tools for making spatio-temporal controlled transgenic mice in the pronuclear injection steps of these transgenes into the fertilized eggs.