Development of a tightly regulated U6 promoter for shRNA expression

Short hairpin RNAs (shRNAs) have been used to achieve stable target knockdown in a variety of biological systems. Here, we report the development of a tightly regulated tetracycline-responsive human U6 promoter for shRNA expression. By engineering two copies of the tet operators flanking the TATA box of the human U6 promoter, we created a U6 promoter derivative (202) that exhibited much lower basal transcriptional activity compared with recently reported inducible pol III dependent promoters. As a consequence of its tighter regulation, the 202 system greatly improved the success rate in making inducible knockdown cell lines. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.