title: Lentiviral vectors encoding tetracycline-dependent repressors and transactivators for reversible knockdown of gene expression: a comparative study.
abstract: BACKGROUND
  RNA interference (RNAi)-mediated by the expression of short hairpin RNAs (shRNAs) has emerged as a powerful experimental tool for reverse genetic studies in mammalian cells. A number of recent reports have described approaches allowing regulated production of shRNAs based on modified RNA polymerase II (Pol II) or RNA polymerase III (Pol III) promoters, controlled by drug-responsive transactivators or repressors such as tetracycline (Tet)-dependent transactivators and repressors. However, the usefulness of these approaches is often times limited, caused by inefficient delivery and/or expression of shRNA-encoding sequences in target cells and/or poor design of shRNAs sequences. With a view toward optimizing Tet-regulated shRNA expression in mammalian cells, we compared the capacity of a variety of hybrid Pol III promoters to express short shRNAs in target cells following lentivirus-mediated delivery of shRNA-encoding cassettes.
  
  
  RESULTS
  RNAi-mediated knockdown of gene expression in target cells, controlled by a modified Tet-repressor (TetR) in the presence of doxycycline (Dox) was robust. Expression of shRNAs from engineered human U6 (hU6) promoters containing a single tetracycline operator (TO) sequence between the proximal sequence element (PSE) and the TATA box, or an improved second-generation Tet-responsive promoter element (TRE) placed upstream of the promoter was tight and reversible as judged using quantitative protein measurements. We also established and tested a novel hU6 promoter system in which the distal sequence element (DSE) of the hU6 promoter was replaced with a second-generation TRE. In this system, positive regulation of shRNA production is mediated by novel Tet-dependent transactivators bearing transactivation domains derived from the human Sp1 transcription factor.
  
  
  CONCLUSIONS
  Our modified lentiviral vector system resulted in tight and reversible knockdown of target gene expression in unsorted cell populations. Tightly regulated target gene knockdown was observed with vectors containing either a single TO sequence or a second-generation TRE using carefully controlled transduction conditions. We expect these vectors to ultimately find applications for tight and reversible RNAi in mammalian cells in vivo.
authors.affiliation: Gene Therapy Program, Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA. plutak@gmail.com <plutak@gmail.com>
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Pluta
authors.forename: William
authors.initials: W
authors.lastname: Diehl
authors.forename: Xian-Yang
authors.initials: XY
authors.lastname: Zhang
authors.forename: Robert
authors.initials: R
authors.lastname: Kutner
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Bialkowska
authors.forename: Jakob
authors.initials: J
authors.lastname: Reiser
mesh_terms: D015870:Gene Expression / Q000235:genetics*; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D013752:Tetracycline; D015534:Trans-Activators / Q000235:genetics*; D014162:Transfection / Q000379:methods
chemical_list: D012097:Repressor Proteins; D015534:Trans-Activators; D013752:Tetracycline