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|Title:||Identification and characterization of IFI30 as a glioblastoma specific promoter for glioma gene therapy||Authors:||MADHUMITHA RENGASAMY||Keywords:||IFI30, Glioblastoma, Gene therapy, Baculovirus, HSVTk||Issue Date:||30-Apr-2009||Citation:||MADHUMITHA RENGASAMY (2009-04-30). Identification and characterization of IFI30 as a glioblastoma specific promoter for glioma gene therapy. ScholarBank@NUS Repository.||Abstract:||Transcriptional targeting involves the use of tissue-specific or tumour-specific promoters to drive gene expression selectively in cancer cells. Though substantial work has been done in transcriptional targeting of hepatoma and breast cancer, little work has been done in the characterization of a glioblastoma specific promoter.
This study deals with the identification, isolation and characterization of a glioblastoma specific promoter for suicide gene therapy. The gene coding for IFI30, interferon gamma-inducible protein 30 (also known as GILT) was found to be up regulated in glioblastoma with respect to normal brain tissue through Microarray and Real Time PCR analyses.
The 2 kb region upstream of the transcriptional start site of IFI30 was then taken to check for promoter activity. Promoter activity was studied through luciferase assays in glioma cell lines, normal human astrocytes and other
cell lines including breast and cervical cancer cell lines. Deletion analysis was carried out to find the shortest fragment of the promoter that showed high activity in glioma cell lines and minimal activity in normal human
The 0.68 kb fragment of the IFI30 promoter was chosen to drive expression of the suicide gene-Herpes Simplex Virus Thymidine kinase (HSV-Tk). This IFI30-HSVTk construct was used in the generation of recombinant baculoviruses (BV-IFI30-HSVTk). U87 (glioma cell line) and NHA (normal human astrocytes) cells were transduced with BV-IFI30-HSVTk and subjected to ganciclovir (GCV) treatment. Thirty-six hours after the addition of 50&181;M GCV, BV-IFI30-HSVTk was able to kill 67% of the U87 cells with minimal toxicity to NHA.
To confirm that cell death was due to expression of the HSV-Tk protein, a western blot assay was performed using whole cell protein from transduced NHA and U87 cells. As expected, there was high expression of the HSV-Tk protein in U87 cells and minimal expression in NHA.
This is the first study that characterizes the use of the IFI30 promoter for transcriptional targeting in glioma and paves way for further research in the use of this promoter for in-vivo therapy.
|Appears in Collections:||Master's Theses (Open)|
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