HIV-specific transgene expression via progressively truncated Tat-Responsive LTR promoters

ORCiD

Nejat Düzgüneş: 0000-0001-6159-1391

Department

Biomedical Sciences

Document Type

Conference Presentation

Conference Title

International Association for Dental Research (IADR)/Annual Meeting of the American Association for Dental Research (AADR) General Session and Exhibition

Location

San Diego, CA

Conference Dates

March 16-19, 2011

Date of Presentation

3-17-2011

Abstract

Objective: Current anti-retroviral therapies against HIV infection are unable to eradicate the chromosomally integrated pro-viral genome. This study seeks to develop a promoter element that is responsive to the HIV transcriptional activator Tat, but not cellular transcription factors. This HIV-specific promoter may be used to drive the expression of suicide genes that would induce cell death specifically in HIV-infected cells. Methods: The HIV-LTR-tar (promoter) region was generated by gene synthesis. Five progressively truncated clones were generated using PCR and inserted into a luciferase-expressing vector and co-transfected into HeLa cells with a plasmid expressing Tat (pHXBDbgl), using Metafectene. Basic vector PGL-3 or herring sperm DNA were used as negative controls. Luciferase expression driven by the modified tar allowed us to determine the region of the LTR promoter most specifically responsive to Tat, and not to other cellular transcription factors. Results: Luciferase activity in cells transfected with both LTR4 and pHXBDbgl was 12,137 ± 914 RLU/mL, while that in control cells transfected with LTR4 and basic vector (no pHXBDbgl) was 204 ± 4 RLU/mL. In cells transfected with LTR1, LTR2, or LTR3, luciferase expression was relatively higher in the presence and absence of pHXBDbgl. In cells containing LTR5 and LTR6 luciferase expression was generally lower compared to LTR4 in the presence and absence of pHXBDbgl. This indicates a 60-fold increase in Tat-specific expression. Conclusions: Higher luciferase expression in cells containing LTR1, LTR2, and LTR3 probably resulted from non-Tat-specific transcriptional activating regions contained on those fragments of the HIV LTR-tar promoter region. LTR4 is the HIV transcriptional activating region most specific to, and yielding the highest gene expression, by Tat activation. Therefore, LTR4 is a candidate for specific activation of suicide genes in HIV-infected cells.

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