Development of a Tetracycline Inducible Retroviral RNAi Vector Using the Mutated TATA Box Form of the U6 Promoter and the Mutant TATA Box Binding Protein

Abstract

The development of systems for inducible siRNA expression can give us an efficient tool to examine loss-of-function phenotypes in a cell of interest without the need to consider lethality or epigenetic adaptation of cells. For this purpose, we constructed the retroviral RNAi vector containing the modified U6 promoter. In this vector, the U6 promoter of RNA Pol III is regulated by tetracycline and allows the inducible knockdown of gene of interest by siRNA. To regulate RNAi expression, we used the mouse U6 promoter that contains the modified TATA-box and the mutant TATA box binding protein which specifically binds to the modified U6 promoter. The retroviral backbone used in this vector provides the applicable potential in a wide variety of models including primary cells, terminally differentiated cells and ES cells. The inducible expression of siRNA in this vector was tested and confirmed by observing the significant downregulation of the exogenous EGFP expression in the presence of tetracycline derivative (doxycycline). Furthermore, using a siRNA plasmid directed against STAT3, we demonstrated the significantly decreased level of the endogenously expressed STAT3 protein by doxycycline treatment.