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.
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Acknowledgments
This work was supported by Istituto Superiore di Sanità, National Research Project on AIDS (grants 40D.14 and 40D.62, ELVIS Italian Network on LTNP), and EC Project number LSHP-CT-2007-037616 (Genetic and Immunological Studies of European and African HIV-1+ Long Term Non-Progressors, GISHEAL) (to C. Casoli).
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Bignami, F., Sozzi, R.A., Pilotti, E. (2017). Generation of a Tet-On Expression System to Study Transactivation Ability of Tax-2. In: Casoli, C. (eds) Human T-Lymphotropic Viruses. Methods in Molecular Biology, vol 1582. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6872-5_7
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DOI: https://doi.org/10.1007/978-1-4939-6872-5_7
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