Generation of lentiviral-mediated shRNA vectors to inhibit the expression of caspase-1 and Pycard/ASC in dendritic cells.

• Main Author: Tan, Bee Kim.
• Other Authors: School of Biological Sciences
• Format: Final Year Project
• Language: English
• Published: 2010
• Subjects: DRNTU::Science::Biological sciences::Microbiology::Immunology
• Online Access: http://hdl.handle.net/10356/39450
• Institution: Nanyang Technological University

Dendritic cells (DC) originate from hematopoietic precursors and they detect endogenous and exogenous stimuli which lead to an inflammatory response. The “inflammasome” is a large protein complex which consists of Caspase-1 and Pycard/ASC which are involved in IL-1β and IL-18 processing. However, the role of DC in the recognition of these factors remains poorly understood. Recently, lentiviral vectors represented a tool to achieve a transient mode of gene-delivery to silence genes in hematopoetic cells. Here, we generated a third generation lentiviral vector system to deliver short-hairpin RNA (shRNA) to silence caspase-1 and Pycard/ASC in growth-factor-dependent murine DC (D1 cells). The generated shRNAs were successfully confirmed and cloned into lentiviral vectors for lentiviral transfection in HEK 293T cells. We observed that HeLa cells used for determining viral titers were not receptive to lentiviral transduction. On the contrary, transduced D1 cells demonstrated a high LNGFR+ expression profile ranging from 40% to 90% transduction efficiencies at 30 MOI. Collectively, we have generated an efficient lentiviral vector-mediated shRNA silencing system in primary cell lines which could provide an insight to into the biological mechanisms involved in inflammasome activation.