Project

Engineering a cell line for analysis of Type I interferon signaling.

Controlling interferon production will reduce the frequency of relapses of multiple sclerosis and provide a better quality of life for patients. Finding the gatekeepers within the pDC pathway that controls IFN production will lead to therapeutic developments for 2.3 million MS patients. The goal of this project was to construct two cell lines constitutively expressing TLR-9 and MyD88 with the c-terminal GFP tag to elucidate the involvement of the receptor X signaling and the IFN-α transcription pathway. This involved, 1) designing primers with att flanking sites to use the 2- fragment Gateway® recombination system, 2) delivering the recombinant DNA into the HEK293 cells, and 3) perform an impact analysis of final product as an in vitro model of the mouse pDC signaling pathway. As a result of this project there are two engineered cell lines that constitutively express TLR9-GFP and MyD88-GFP. The GFP reporter tag attached to each gene of interest provided a way to visualize the intracellular patterns of protein localization through microscopy. Anti-GFP antibody was used to western blot the engineered cells and detected higher levels of MyD88 protein expression compared to TLR-9 expression. When MyD88-GFP and TLR9-GFP cells were stimulated with receptor X agonist and antagonist there was no change in protein expression or localization patterns suggesting they may not be the gatekeepers to controlling IFN production. Insertion of GFP tag does not alter protein folding or expression of TLR-9 or MyD88. The engineered cell lines can be used to reconstitute the pDC pathway and has proven to survive facile manipulations.

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