Identification of cellular targets for biologically active molecules
AssigneeIrm LLC
InventorsJeremy S. Caldwell, Sumit K. Chanda, Nikunj V. Somia, John B. Hogenesch, Michael P. Cooke, Pedro Aza-Blanc
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Abstract
A genetic screening methodology for rapid identification of candidate targets of any small molecule cellular effectors and other signals and modulators of cellular functions and pathways is provided. The effect of a small molecule or other signal on a cell is titrated by expressing within the cell cDNA that encodes a polypeptide that is the molecular target or that is responsible for directly or indirectly producing the molecular target.
Claims122
A method for identifying a function of endogenous gene by modulating the level of a product encoded by the endogenous gene, the method comprising:
- a) introducing nucleic acid molecules into populations of reporter cells to form an addressable collection of cell populations, wherein cells of a first cell population comprise a different introduced nucleic acid from cells of at least a second cell population and
- b) identifying cell populations in the collection in which cells exhibit a phenotype that is different in the presence of the introduced nucleic acid molecule from the phenotype exhibited in its absence, thereby identifying a nucleic acid molecule that modulates the level of a product of an endogenous gene or genes that effect the phenotype and identifying the function of the endogenous gene or genes.
A method for identifying the targets of a perturbagen by modulating the level of an endogenous messenger RNA, comprising:
- a) introducing a nucleic acid molecule into populations of reporter cells to form an addressable collection of cell populations, wherein cells of a first cell population comprise a different introduced nucleic acid from cells of at least a second cell population;
- and
- b) exposing the cells to a perturbagen that potentially alters a phenotype;
- and
- c) identifying cell populations in the collection in which cells exhibit a phenotype that is different in the presence of the introduced nucleic acid molecule and the perturbagen compared to the phenotype exhibited by the cells in the absence of the introduced nucleic acid molecule and the perturbagen;
- wherein a) and b) are performed either simultaneously or sequentially in either order, and the method thereby identifies a target or targets of the perturbagen.
A method of identifying cDNA that, when expressed in a cell, causes an altered response of the cell to a biologically active molecule compared to a control cell, the method comprising:
- (a) providing a plurality of reporter cells that each comprises the cell and a construct that comprises nucleic acid encoding a product operably linked to a promoter such that the cDNA is expressed in the reporter cell, wherein different nucleic acid molecules are expressed in each of the plurality of reporter cells;
- (b) contacting the each of plurality of reporter cells with a biologically active molecule or expositing the cells to a condition that alters gene expression;
- and
- (c) identifying any reporter cells that have an altered response to the biologically active molecule or the condition compared to a control.
A combination, comprising:
- a) an addressable collection of reporter cells, wherein:
- the reporter cells generate an output representative of expression of a gene or a cellular activity;
- and the reporter cells comprise a promoter operatively linked to a reporter gene;
- and
- b) a library of nucleic acid molecules.
A method for identifying the target of an effector or a target for an effector of gene expression or for a cellular activity, comprising:
- a) providing an addressable collection of reporter cells, wherein the reporter cells generate an output representative of expression of the gene or the cellular activity;
- b) contacting the cells with an effector of the activity or expression;
- c) introducing nucleic acid encoding a potential target of the effector, wherein the contacting and introducing step are performed either simultaneously or sequentially in either order;
- and
- d) identifying cells in the collection that exhibit expression or activity that is different in the presence of the nucleic acid than in its absence, thereby identifying the target of or for an effector of gene expression or a cellular activity.
Dependencies are inferred from the claim text.