CELL BASED SENSOR

摘要

Cell based sensor. The present invention relates to a novel cell based sensor useful for drug discovery that comprises a cell line with professional regulated exocytosis of secretory granules transfected with a non-protease hydrolase as a reporter polypeptide stored in the regulated secretory granules of the cell line with professional regulated exocytosis and having either an endogenous or a heterologous molecule as a modulator of regulated secretory granules exocytosis. Said granule stored non protease-hydrolase reporter having at least: a high resistance to conditions already present inside the granules such as low pH and proteolysis by other proteases; enzymatic activity after exocytosis; a highly specific substrate; absence of toxicity when cell thawing; a very low level of secretion under unstimulated or basal conditions; and a high signal to background activity in a media compatible with cell culture viability and granule exocytosis for a high throughput robust and sensitive detection.

主权项

1. A cell based sensor that comprises:
a. A hematopoietic cell line with regulated exocytosis of secretory granules; b. A granule stored non-protease hydrolase reporter transfected into the cell line of (a) and overexpressed under the control of either a strong constitutive promoter or a strong inducible promoter; c. An endogenous modulator or a transfected heterologous modulator of regulated exocytosis of the secretory granules of the cell line of (a); d. A cell impermeable substrate selected from the group comprising: a colorimetric, a fluorescent or a luminescent substrate specific for detection of a secreted non-protease hydrolase activity;which allows measuring the effect of a specific ligand on the modulator of regulated exocytosis.