FACS screening assay

One way of identifying novel interactions is to screen complex prey cDNA libraries. We have developed a cell line that exhibits three features making it suitable for this purpose. First, the cell line is derived from the HEK293 FlpIn T-Rex cell line (Invitrogen), which allows for the rapid generation of isogenic pools of bait-expressing cells through recombinase-assisted integration of a chimeric bait receptor construct. Second, it expresses the mouse ecotropic receptor, allowing for the entry of the retroviral prey cDNA library. Third, it harbors a reporter cassette consisting of a STAT3-responsive rPAP1 promoter-driven hIL5Rα-derived membrane tag.

The screening protocol starts with retroviral delivery of the prey cDNA library to the bait-expressing cell pool. After stimulation with the appropriate ligand, positive cells containing a prey that interacts with the bait are enriched using magnetobeads that attach to the hIL5Rα membrane tag. In a next step, false positive cells are sorted out with a cell sorter. Finally, cells are again stimulated with ligand and single positive cells are sorted into multiwell plates. The clones are validated using a dot blot assay and the identity of the prey is determined through RT-PCR and sequencing.

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