Mammalian two-hybrid system for screening for modulators of the accumulation of metabolic products
First Claim
1. A method of screening for an agent that modulates the ability of a cell to accumulate or to degrade a metabolic product, said method comprising:
- (i) providing a mammalian cell comprising;
a nucleic acid encoding a peptide binding site and an effector gene;
a first chimeric protein comprising a nucleic acid binding domain that binds said peptide binding site attached to said metabolic product or to a ligand that binds to said metabolic product; and
a second chimeric protein comprising an expression control protein attached to said metabolic product or to said ligand that binds to said metabolic product such that when said first chimeric protein comprises said metabolic product, said second chimeric protein comprises said ligand and when said first chimeric protein comprises said ligand, said second chimeric protein comprises said metabolic product;
(ii) contacting said cell with a test agent; and
(iii) detecting an alteration of expression of said effector gene wherein a difference in the expression of said effector gene in said test cell, as compared to a control cell contacted with a lower concentration of test agent or no test agent indicates that said test agent modulates the ability of said cell to accumulate or degrade said metabolic product, wherein said mammalian cell comprises a degradation deficiency of the metabolic product.
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Abstract
This invention utilizes a two-hybrid system to screen for agents that modulate the ability of a cell to degrade or to accumulate a metabolic product or to selective kill a cell or to selectively express a gene or cDNA in a cell that has a defect in its ability to degrade or to accumulate a metabolic product. One embodiment provides a mammalian cell comprising a nucleic acid encoding a peptide binding domain and an effector gene; a first chimeric protein comprising a nucleic acid binding domain that binds the peptide binding domain attached to the metabolic product or to a ligand that binds to the metabolic product; and a second chimeric protein comprising an expression control protein attached to the metabolic product or to the ligand that binds to the metabolic product such that when the first chimeric protein comprises the metabolic product, the second chimeric protein comprises the ligand and when the first chimeric protein comprises the ligand, the second chimeric protein comprises the metabolic product. The cell is contacted with a test agent and in alteration of expression of the effector gene is detected (if present) where a change in expression of the effector gene in response to the test agent indicates that the test agent modulates the ability of the cell to accumulate or degrade the metabolic product.
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Citations
20 Claims
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1. A method of screening for an agent that modulates the ability of a cell to accumulate or to degrade a metabolic product, said method comprising:
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(i) providing a mammalian cell comprising;
a nucleic acid encoding a peptide binding site and an effector gene;
a first chimeric protein comprising a nucleic acid binding domain that binds said peptide binding site attached to said metabolic product or to a ligand that binds to said metabolic product; and
a second chimeric protein comprising an expression control protein attached to said metabolic product or to said ligand that binds to said metabolic product such that when said first chimeric protein comprises said metabolic product, said second chimeric protein comprises said ligand and when said first chimeric protein comprises said ligand, said second chimeric protein comprises said metabolic product;
(ii) contacting said cell with a test agent; and
(iii) detecting an alteration of expression of said effector gene wherein a difference in the expression of said effector gene in said test cell, as compared to a control cell contacted with a lower concentration of test agent or no test agent indicates that said test agent modulates the ability of said cell to accumulate or degrade said metabolic product, wherein said mammalian cell comprises a degradation deficiency of the metabolic product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
said nucleic acid encodes a GAL-4 binding site, and said effector gene is a reporter gene; said first chimeric protein comprises a GAL-4 nucleic acid binding protein and a beta catenin or a Tcf;
said second chimeric protein comprises a VP-16 and beta catenin or a Tcf.
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18. The method of claim 17, wherein said Tcf is Tcf4.
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19. The method of claim 1, wherein said cell comprises a nucleic acid encoding said first or said second chimeric protein under control of a tissue specific or an inducible promoter.
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20. The method of claim 19, wherein said promoter is an ecdysone promoter.
Specification