Chapter title |
Generation of a Tet-On Expression System to Study Transactivation Ability of Tax-2
|
---|---|
Chapter number | 7 |
Book title |
Human T-Lymphotropic Viruses
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6872-5_7 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6870-1, 978-1-4939-6872-5
|
Authors |
Fabio Bignami, Riccardo Alessio Sozzi, Elisabetta Pilotti, Bignami, Fabio, Sozzi, Riccardo Alessio, Pilotti, Elisabetta |
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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