title: Generation of a Tet-On Expression System to Study Transactivation Ability of Tax-2.
abstract: HTLV Tax proteins (Tax-1 and Tax-2) are known to be able to transactivate several host cellular genes involved in complex molecular pathways. Here, we describe a stable and regulated high-level expression model based on Tet-On system, to study the capacity of Tax-2 to transactivate host genes. In particular, the Jurkat Tet-On cell line suitable for evaluating the ability of Tax-2 to stimulate transactivation of a specific host gene, CCL3L1 (C-C motif chemokine ligand 3 like 1 gene), was selected. Then, a plasmid expressing tax-2 gene under control of a tetracycline-response element was constructed. To avoid the production of a fusion protein between the report gene and the inserted gene, a bidirectional plasmid was designed. Maximum expression and fast response time were achieved by using nucleofection technology as transfection method. After developing an optimized protocol for efficiently transferring tax-2 gene in Jurkat Tet-On cellular model and exposing transfected cells to Dox (doxycycline, a tetracycline derivate), a kinetics of tax-2 expression through TaqMan Real-time PCR assay was determined.
authors.affiliation: Center for Medical Research and Molecular Diagnostic, GEMIB Laboratory, Borgo Girolamo Cantelli 11, 43121, Parma, PR, Italy.|Department of Clinical Sciences, University of Milan, Milan, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Bignami
authors.affiliation: Department of Environmental Sciences, University of Parma, Parma, Italy.
authors.forename: Riccardo Alessio
authors.initials: RA
authors.lastname: Sozzi
authors.affiliation: Center for Medical Research and Molecular Diagnostic, GEMIB Laboratory, Borgo Girolamo Cantelli 11, 43121, Parma, PR, Italy. elisabetta.pilotti@gmail.com.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Pilotti
mesh_terms: D019742:Chemokines, CC / Q000096:biosynthesis / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D016356:Gene Products, tax / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D019169:Jurkat Cells; D015533:Transcriptional Activation / Q000187:drug effects*
chemical_list: C492767:CCL3L1 protein, human; D019742:Chemokines, CC; D016356:Gene Products, tax; C000611828:tax2 protein, Human T-lymphotrophic virus 2; D004318:Doxycycline
keywords: 2-ΔΔCT method; Bidirectional vectors; HTLV Tax-2; Nucleofection; TaqMan Real-Time PCR; Tet-On systems