Functional genomics using zinc finger proteins
First Claim
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1. A method for establishing an association between a gene and a selected phenotype in a cell, the method comprising the steps of:
- (i) identifying a plurality of candidate genes, wherein at least one of the candidate genes is not operably linked to heterologous sequences;
(ii) providing a first engineered zinc finger protein that binds to a first target site of a first candidate gene;
(iii) culturing a first cell under conditions where the first zinc finger protein contacts the first candidate gene, wherein the first zinc finger protein modulates expression of the first candidate gene;
(iv) determining the expression pattern of the candidate genes and determining whether or not the first candidate gene is associated with the selected phenotype; and
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(v) repeating steps (ii)-(v) for each candidate gene.
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Abstract
The present invention provides methods of regulating gene expression using recombinant zinc finger proteins, for functional genomics and target validation applications.
313 Citations
55 Claims
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1. A method for establishing an association between a gene and a selected phenotype in a cell, the method comprising the steps of:
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(i) identifying a plurality of candidate genes, wherein at least one of the candidate genes is not operably linked to heterologous sequences;
(ii) providing a first engineered zinc finger protein that binds to a first target site of a first candidate gene;
(iii) culturing a first cell under conditions where the first zinc finger protein contacts the first candidate gene, wherein the first zinc finger protein modulates expression of the first candidate gene;
(iv) determining the expression pattern of the candidate genes and determining whether or not the first candidate gene is associated with the selected phenotype; and
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(v) repeating steps (ii)-(v) for each candidate gene. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A method for establishing an association between a gene and a selected phenotype in a cell, the method comprising the steps of:
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(i) selecting a first candidate gene, wherein the first candidate gene is not operably linked to heterologous sequences;
(ii) providing a first engineered zinc finger protein that binds to a target site of the first candidate gene and second zinc finger protein that binds to a second target site of the first candidate gene;
(iii) culturing a first cell under conditions where the first zinc finger protein contacts the first candidate gene, and culturing a second cell under conditions where the second zinc finger protein contacts the first candidate gene, wherein the first and the second zinc finger proteins modulate expression of the first candidate gene; and
(iv) assaying the first and second cells for a selected phenotype, thereby identifying whether or not the first candidate gene is associated with the selected phenotype. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54)
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55. A method for establishing an association between a gene and a selected phenotype in a cell, the method comprising the steps of:
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(i) selecting a first candidate gene, wherein the first candidate gene is not operably linked to heterologous sequences;
(ii) providing a first engineered zinc finger protein that binds to a first target site of the first candidate gene;
(iii) culturing a first cell under conditions where the first zinc finger protein contacts the first candidate gene, wherein the first zinc finger protein modulates expression of the first candidate gene; and
(iv) assaying for a selected phenotype, thereby identifying whether or not the first candidate gene is associated with the selected phenotype.
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Specification