Abstract
Regulated gene expression will provide important platforms from which gene functions can be investigated and safer means of gene therapy may be developed. Histone deacetylases have recently been shown to play an important role in regulating gene expression. Here we investigated whether a more tightly controlled expression could be achieved by using a novel chimeric repressor that recruits histone deacetylases to a tetracycline-responsive promoter. This chimeric repressor was engineered by fusing the tetracycline repressor (TetR) with an mSin3-interacting domain of human Mad1 and was shown to bind the tetO(2) element with high affinity, and its binding was efficiently abrogated by doxycycline. The chimeric repressor was shown to directly interact with mSin3 of the histone deacetylase complex. This inducible system was further simplified by using a single vector that contained both a chimeric repressor expression cassette and a tetracycline-responsive promoter. When transiently introduced into mammalian cells, the chimeric repressor system exhibited a significantly lower basal level of luciferase activity (up to 25-fold) than that of the TetR control. When stably transfected into HEK 293 cells, the chimeric repressor system was shown to exert a tight control of green fluorescent protein expression in a doxycycline dose- and time-dependent fashion. Therefore, this novel chimeric repressor provides an effective means for more tightly regulated gene expression, and the simplified inducible system may be used for a broad range of basic and clinical studies.
Highlights
Regulated gene expression will provide important platforms from which gene functions can be investigated and safer means of gene therapy may be developed
This chimeric repressor was engineered by fusing the tetracycline repressor (TetR) with an mSin3-interacting domain of human Mad1 and was shown to bind the tetO2 element with high affinity, and its binding was efficiently abrogated by doxycycline
We engineered a tightly controlled gene expression system mediated by a chimeric repressor that recruits histone deacetylases
Summary
Cell Culture, Medium, and Chemicals—Human embryonic kidney cell line HEK 293, human colon cancer line HCT116, and human osteosarcoma line 143B were purchased from the American Type Culture Collection (Manassas, VA). 293 cells were maintained in Dulbecco’s modified Eagle’s medium (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Mediatech), 100 units/ml penicillin, and 100 g/ml streptomycin at 37 °C in 5% CO2. Two oligonucleotides were denatured, annealed, and extended with Platinum Taq (Life Technologies, Inc.) for one cycle to generate a cassette that was subsequently cloned downstream to a CMV promoter, resulting in pCMV-TO4 In this vector, the first tetO2 site was precisely positioned 10 nucleotides downstream from the TATA box of the CMV promoter (Fig. 1B). Each DNA binding assay was supplemented with 0.1– 0.2 g of GST fusion proteins; a nonspecific competitor, poly(dI-dC) (6 g/ml); and 0.5 ng of 32P-labeled probe (Ϸ106 dpm). GST Fusion Protein Pull-down Assay and Western Blotting Analysis—Exponentially growing 293 cells were collected and lysed in a cell lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.5% Nonidet P-40) containing protease inhibitors (Roche Molecular Biochemicals). Establishment of Stable Inducible Lines Using pTHE-GFP—HEK 293 cells were plated in a 25-cm flask and transfected with 2 g of PacI-linearized pTHE-GFP plasmid DNA by using LipofectAMINE
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
