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 (2O2) that exhibited much lower basal transcriptional activity compared with recently reported inducible pol III dependent promoters. As a consequence of its tighter regulation, the 2O2 system greatly improved the success rate in making inducible knockdown cell lines.
Lin
Xiaoyu
X
Cancer Research, Global Pharmaceutical Research and Development, AP10/L01, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA.
Yang
Jianguo
J
Chen
Jun
J
Gunasekera
Angelo
A
Fesik
Stephen W
SW
Shen
Yu
Y
D015153:Blotting, Western; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D014644:Genetic Variation; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box; D013752:Tetracycline / Q000494:pharmacology; D014158:Transcription, Genetic
D034741:RNA, Small Interfering; D008156:Luciferases; D013752:Tetracycline