Mammalian two-hybrid system for screening for modulators of the accumulation of metabolic products
First Claim
1. A cell comprising:
- an accumulation or degradation of a metabolic product;
a nucleic acid encoding a peptide binding site and an effector gene;
a first chimeric protein comprising a nucleic acid binding protein that binds said peptide binding site where said nucleic acid binding protein is 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;
wherein said cell is a cell selected from the group consisting of SW480, SW48, DLD-1, HCT-116,HT29, 293, U-20S, T-47D, MCF-7, HeLa, A549, Hep G2, a mammalian cell, and a Jarkat cell.
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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 an 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.
12 Citations
28 Claims
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1. A cell comprising:
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an accumulation or degradation of a metabolic product; a nucleic acid encoding a peptide binding site and an effector gene; a first chimeric protein comprising a nucleic acid binding protein that binds said peptide binding site where said nucleic acid binding protein is 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; wherein said cell is a cell selected from the group consisting of SW480, SW48, DLD-1, HCT-116,HT29, 293, U-20S, T-47D, MCF-7, HeLa, A549, Hep G2, a mammalian cell, and a Jarkat cell. - View Dependent Claims (2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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4. A cell comprising:
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an accumulation or degradation of a metabolic product; a nucleic acid encoding a peptide binding site and an effector gene; a first chimeric protein comprising a nucleic acid binding protein that binds said peptide binding site where said nucleic acid binding protein is 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; wherein said expression control protein is a repressor.
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16. A nucleic acid comprising a nucleic acid selected from the group consisting of
a nucleic acid encoding a chimeric protein comprising a nucleic acid binding domain attached to a Tcf4 or to a beta catenin, and a nucleic acid encoding a transactivator attached to a beta catenin or to a Tcf4.
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23. A vector comprising:
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a nucleic acid encoding a peptide binding site and an effector gene; a nucleic acid encoding a first chimeric protein comprising a nucleic acid binding protein that binds said peptide binding site where said nucleic acid binding protein is attached to said metabolic product or to a ligand that binds to said metabolic product; and a nucleic acid encoding 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; wherein said a nucleic acid encoding a first chimeric protein further encodes an HIV TAT domain attached to said first chimeric protein or; wherein said a nucleic acid encoding a second chimeric protein further encodes an HIV TAT domain attached to said second chimeric protein.
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24. A nucleic acid encoding a first chimeric protein comprising a nucleic acid binding protein attached to:
- a metabolic product or to a ligand that binds to a metabolic product;
wherein said nucleic acid further encodes an HIV TAT domain attached to said first chimeric protein.
- a metabolic product or to a ligand that binds to a metabolic product;
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25. A nucleic acid encoding a second chimeric protein comprising an expression control protein attached to a metabolic product or to a ligand that binds to said metabolic product;
wherein said nucleic acid further encodes an HIV TAT domain attached to said first chimeric protein.
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26. A kit for screening for an agent that modulates the ability of a cell to accumulate or to degrade a metabolic product, said kit comprising:
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a container containing a mammalian cell comprising; a nucleic acid encoding a protein binding site and an effector gene; a first chimeric protein comprising a nucleic acid binding protein that binds said protein binding site attached to said metabolic product or to a ligand that binds to said metabolic product; 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.
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Specification