Development of a tetracycline-inducible lentiviral vector with an instant regulatory system : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science (MSc) in Biochemistry at Massey University, Manawatu, New Zealand

Abstract

Lentiviral vectors, originally derived from human immunodeficiency virus, provide highly efficient viral gene delivery vehicles. Lentiviral vectors often use a constitutive promoter to drive the expression of a therapeutic gene. To regulate the expression of a therapeutic gene, a regulatory system such as Tet-On needs to be established in the target cell lines to produce a regulatory protein, reverse Tet-responsive transcriptional activator (rtTA). The expressed rtTA binds to the tetracycline responsive element (TRE) in the promoter in response to doxycycline and activates transcription of gene of interest. A hypothesis in this study is based on the speculation that a basal leaky expression of rtTA in the bi-directional TRE vectors allows instantly inducible expression of a gene of interest and thereby avoids the time-consuming procedures for generating Tet-On cell lines. Based on this hypothesis, a novel lentiviral vector has been developed to examine an instant induction of PP2Cβ as a target gene. Three instantly inducible bicistronic lentiviral vectors [pLenti-Bi-TRE-Tet-on (V), pLenti-Bi-TRE-Tet-on-PP2Cβ WT (WT), pLenti- Bi-TRE-Tet-on-PP2Cβ MUT (MUT)] were constructed and characterised to assess the usefulness of these vectors. Transient transfection of both WT and MUT vectors into HEK293T cells showed a great induction of PP2Cβ expression upon 24 h of 1 μM doxycycline treatment. The result promises the use of these vectors as a mammalian expression plasmid with a feature of inducible target gene expression. However, viral infection studies involving lentiviral packaging and infection procedures did not show a reproducible expression of rtTA or PP2Cβ in HEK293T cells. Therefore, the inducibility of viral transduction needs to be improved for the future studies of PP2Cβ in primary cells.