Two Stable H4 Neuroglioma Cell Lines Expressing Alpha-synuclein Bioluminescent- and Fluorescent- Protein Complemention Pairs

Technology #2013-134

A plasmid was constructed to contain an FRT cassette, hygromycin resistance, and a tetracycline-driven bidirectional promoter. Then the cDNA encoding alpha-synuclein protein was fused with either the N- or C-terminal half of gaussia luciferase, or with the N- or C-terminal half of Yellow Fluorescent Protein sequences. These fusion constructs were then inserted in the bidirectional plasmid previously made. Using the Flp In system and a tet-off human neuroglioma cell line, both bidirectional vectors were used to generate two stable and tetracycline-regulatible cell lines; one expressing the alpha-synuclein-gaussia luciferase halves and the other one expressing the alpha-synuclein-YFP halves. When alpha-synuclein -alpha-synuclein interactions occur in these stable cell lines the N- and C-terminal halves of the gaussia luciferase or the YFP are brought close together thus reconstituting luciferase enzymatic activity or YFP fluorescence. Consequently, the bioluminescence or fluorescence can be monitored as surrogate markers for alpha-synuclein oligomer formation and can be directly monitored using a microscope or a plate reader. Taken together, we developed novel cell lines based on the concept of protein complementation to monitor alpha-synuclein oligomerization. The cell lines can be used for high throughput screening applications for modulators of alpha-synuclein oligomerization.