title: Development of a doxycycline-inducible lentiviral plasmid with an instant regulatory feature.
abstract: Lentiviruses provide highly efficient gene delivery vehicles in both dividing and non-dividing cells. Inducible gene expression systems often employ a specific cell line that constitutively expresses a regulatory protein for transgene expression. As one of such inducible expression systems the Tet-On system uses a cell line expressing reverse tetracycline-responsive transcriptional activator (rtTA). The rtTA protein binds to the tetracycline-responsive element (TRE) in the promoter and activates transcription of a transgene in a doxycycline-dependent manner. To establish a universal and instant regulatory system without generating Tet-On cell lines, the cDNAs of rtTA and a testing target gene (PPM1B) were cloned in the bi-directional TRE-containing promoters. Here, we examined whether a basal leaky expression of rtTA allows instantly inducible expression of both rtTA itself and the target gene, PPM1B in a single plasmid using the two mini-CMV promoters. Transient transfection of the lentiviral plasmids into human embryonic kidney HEK293T cells showed a significant induction of PPM1B expression in response to doxycycline, suggesting that these lentiviral plasmids can be used as an instantly inducible mammalian expression vector. However, the expression of rtTA by lentiviral transduction shows a minimal expression without a consistent response to doxycycline, suggesting that the utility of these lentiviral vectors is limited. A potential solution to overcome lentiviral transgene inactivation is proposed.
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand; Institute of Somatology, Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu 210029, PR China.
authors.forename: Tian
authors.initials: T
authors.lastname: Yang
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
authors.forename: Christopher
authors.initials: C
authors.lastname: Burrows
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand. Electronic address: J.Park@massey.ac.nz.
authors.forename: Jeong Hyeon
authors.initials: JH
authors.lastname: Park
mesh_terms: D003587:Cytomegalovirus / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology*; D015967:Gene Expression Regulation, Viral; D005798:Genes, Bacterial; D005814:Genes, Viral; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D020218:Response Elements; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects*; D014161:Transduction, Genetic
chemical_list: D015534:Trans-Activators; D004318:Doxycycline
keywords: Bicistronic; Doxycycline; Inducible expression; Lentivirus; PPM1B; Tetracycline-responsive element