Array screening assay

To enable rapid screening of a large collection of preys, we developed an arrayed MAPPIT screening assay based on reverse transfection. A set of prey plasmids is mixed with the reporter plasmid and complexed with a transfection agent. These transfection mixtures are arrayed on a solid support, i.e. microtiter plates or microscope slides, and dried. The fact that these prey arrays can be stored for a long time adds to the versatility of the assay platform.

Generation of a cell array. Reverse transfection mixtures containing nucleic acids complexed with a transfection reagent are spotted onto a carrier surface. After drying, these arrays are overlaid with cells and incubated. Cells growing on top of the spots are (reverse) transfected with the nucleic acids present in the dried reverse transfection complexes, resulting in an array of spots containing transfected cells.

To screen a MAPPIT prey array for interaction partners of a specific bait, the plates are overlaid with bait-expressing cells, stimulated with the bait receptor-activating ligand, and assayed for luciferase expression. This simple procedure takes only five days from bait transfection up to luciferase signal measurement, and has been automated to a large extent.

ArrayMAPPIT screening setup. Prey collections are compiled from the human ORFeome collection (CCSB, Boston) using Gateway cloning. A collection of over 12.500 preys (corresponding to the hORFeome v8.1 collection) arrayed in 384well microtiterplates is currently available for screening. In the near future, the assay will be further downsized using microarray printing. 

Since MAPPIT operates in intact mammalian cells and as the array screening protocol is largely automated, parallel screens can be run under different conditions induced by external stimuli that mimic a specific physiological state of the cell (e.g. without/with a ligand that activates a specific signalling cascade, or without/with application of an (a)biotic stress factor). This approach should enable monitoring of dynamic aspects of protein interaction networks.

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