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
dc.contributor.author | Yang, Tian | |
dc.date.accessioned | 2013-05-03T00:20:52Z | |
dc.date.available | 2013-05-03T00:20:52Z | |
dc.date.issued | 2013 | |
dc.description.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. | en |
dc.identifier.uri | http://hdl.handle.net/10179/4319 | |
dc.language.iso | en | en |
dc.publisher | Massey University | en_US |
dc.rights | The Author | en_US |
dc.subject | Lentiviral vectors | en |
dc.subject | Gene therapy | en |
dc.subject | Gene expression | en |
dc.subject | Gene regulation | en |
dc.subject | Tetracycline | en |
dc.subject | Genetic vectors | en |
dc.title | 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 | en |
dc.type | Thesis | en |
massey.contributor.author | Yang, Tian | en |
thesis.degree.discipline | Biochemistry | en |
thesis.degree.grantor | Massey University | en |
thesis.degree.level | Masters | en |
thesis.degree.name | Master of Science (M.Sc.) | en |
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