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US 6,723,531 B2Granted
Method for modulating expression of exogenous genes in mammalian systems, and products related thereto
AssigneeThe Salk Institute for Biological Studies
InventorsRonald M. Evans, David No, Enrique Saez
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Abstract
In accordance with the present invention, there are provided various methods for modulating the expression of an exogenous gene in a mammalian subject employing modified ecdysone receptors. Also provided are modified ecdysone receptors, as well as homomeric and heterodimeric receptors containing same, nucleic acids encoding invention modified ecdysone receptors, modified ecdysone response elements, gene transfer vectors, recombinant cells, and transgenic animals containing nucleic acids encoding invention modified ecdysone receptor.
Claims25
A method for modulating the expression of an exogenous gene in an isolated cell containing:
- (i) a modified ecdysone receptor which, in the presence of a ligand therefor, and optionally in the further presence of a silent partner therefor, binds to a response element, wherein said modified ecdysone receptor comprises:
- (a) a ligand binding domain that binds to an ecdysteroid,
- (b) a DNA-binding domain obtained from a DNA-binding protein, which binds to said response clement;
- and
- (c) an activation domain of a transcription factor, wherein at least one of said DNA-binding domain or said activation domain is not obtained from a native ecdysone receptor, with the proviso that when said activation domain is derived from a glucocorticoid receptor, said DNA-binding domain is not derived from a glucocorticoid receptor or a E. coli LexA protein;
- and
- (ii) a DNA construct comprising said exogenous gene under the control of said response element, wherein said response element:
- (a) is a modified response element which comprises, in any order, a first half-site and a second half-site separated by a spacer of 0-5 nucleotides;
- wherein said first half-site has the sequence:
- —RGBNNM—,
- wherein
- each R is independently selected from A or G;
- each B is independently selected from G, C, or T;
- each N is independently selected from A, T, C, or G;
- and
- each M is independently selected from A or C;
- with the proviso that at least 4 nucleotides of each —RGBNNM—group of nucleotides are identical with the nucleotides at comparable positions of the sequence —AGGTCA—;
- and wherein said second half-site is obtained from a glucocorticoid receptor subfamily response element,
- (b) binds to said modified ecdysone receptor, and
- (c) does not bind to farnesoid X receptor (FXR);
- said method comprising providing to the cell an effective amount of one or more ligands for said modified ecdysone receptor;
- wherein said one or more ligands are not normally present in the cell;
- and wherein said one or more ligands are not toxic to said cell.
20 more elements
A method of inducing the expression of an exogenous gene in an isolated cell containing:
- (i) DNA encoding a modified ecdysone receptor under the control of an inducible promoter, wherein said modified ecdysone receptor, in the presence of a ligand therefor, and optionally in the further presence of a silent partner therefor, binds to a response element, and wherein said modified ecdysone receptor comprises:
- (a) a ligand binding domain that binds to an ecdysteroid,
- (b) a DNA-binding domain obtained from a DNA-binding protein, which binds to said response element;
- and
- (a) an activation domain of a transcription factor,
- wherein at least one of said DNA-binding domain or said activation domain is not obtained from a native ecdysone receptor, with the proviso that when said activation domain is derived from a glucocorticoid receptor, said DNA-binding domain is not derived from a glucocorticoid receptor or an E. coli LexA protein;
- (ii) a DNA construct comprising said exogenous gene under the control of said response element, wherein said response elements;
- (a) is a modified response element which comprises, in any order, a first half-site and a second half-site separated by a spacer of 0-5 nucleotides;
- wherein said first half-site has the sequence:
- —RGBNNM—,
- wherein
- each R is independently selected from A or G;
- each B is independently selected from G, C, or T;
- each N Is independently selected from A, T, C, or G;
- and
- each M is independently selected from A or C;
- with the proviso that at least 4 nucleotides of each —RGBNNM—group of nucleotides are identical with the nucleotides at comparable positions of the sequence —AGGTCA—;
- and wherein said second half-site is obtained from a glucocorticoid receptor subfamily response element,
- (b) binds to said modified ecdysone receptor, and
- (c) does not bind to farnesoid X receptor (FXR);
- and
- (iii) one or more ligands for said modified ecdysone receptor;
- said method comprising subjecting said cell to conditions suitable to induce expression of said modified ecdysone receptor.
20 more elements
A method of inducing expression of an exogenous gene in an isolated cell containing a DNA construct containing said exogenous gene under the control of a response element, wherein said response element:
- (a) is a modified response element which comprises, in any order, a first half-site and a second half-site separated by a spacer of 0-5 nucleotides;
- wherein said first half-site has the sequence;
- —RGBNNM—,
- wherein
- each R is independently selected from A or G;
- each B is independently selected from G, C, or T;
- each N is independently selected from A, T, C, or G;
- and
- each M is independently selected front A or C;
- with thy proviso that at least 4 nucleotides of each —RGBNNM—group of nucleotides are identical with the nucleotides at comparable position of the sequence —AGGTCA—;
- and wherein said second half-site is obtained from a glucocorticoid receptor subfamily response element,
- (b) binds to said modified ecdysone receptor, and
- (c) does not bind to farnesoid X receptor (FXR), said method comprising introducing into said cell;
- a modified ecdysone receptor, wherein said modified ecdysone receptor comprises:
- (a) a ligand binding domain that binds to an ecdysteroid, (b) a DNA-binding domain obtained from a DNA-binding protein, which binds to said response element;
- and (c) an activation domain of a transcription factor, wherein at least one of said DNA-binding domain or said activation domain is not obtained from a native ecdysone receptor, with the proviso that when said activation domain is derived from a glucocorticoid receptor, said DNA-binding domain is not derived from a glucocorticoid receptor or an E. coli LexA protein;
- and
- one or more ligands for said modified ecdysone receptor,
- wherein said receptor, in combination with a ligand therefor, and optionally in the further presence of a silent partner therefor, binds to said response element, activating transcription therefrom.
16 more elements
A method for the expression of a recombinant product detrimental to isolated host cells, said method comprising:
- transforming suitable isolated host cells with:
- (i) DNA encoding a modified ecdysone receptor, wherein said modified ecdysone receptor comprises:
- (a) a ligand binding domain that binds to an ecdysteroid,
- (b) a DNA-binding domain obtained from a DNA-binding protein;
- and
- (c) an activation domain of a transcription factor,
- wherein at least one of said DNA-binding domain or said activation domain is not obtained from a native ecdysone receptor, with the proviso that when said activation domain is derived from a glucocorticoid receptor, said DNA-binding domain is not derived from a glucocorticoid receptor or an E. coli LexA protein;
- and
- (ii) a DNA construct encoding said recombinant product under the control of a response element, wherein said response element;
- (a) is a modified response element which comprises, in any order, a first half-site and a second half-site separated by a spacer of 0-5 nucleotides;
- wherein maid first half-site has the sequence;
- —RGBNNM—,
- wherein
- each R is independently selected from A or G;
- each B is independently selected from G, C, or G;
- each N is independently selected from A, T, C, or G;
- and
- each M is independently selected from A or C;
- with the proviso that at least 4 nucleotides of each —RGBNNM—group of nucleotides are identical with the nucleotides at comparable positions of the sequence —AGGTCA—;
- and wherein said second half-site is obtained from a glucocorticoid receptor subfamily response element
- (b) binds to said modified ecdysone receptor, and
- (c) does not bind to farnesoid X receptor (FXR);
- growing said host cells in suitable media;
- and
- inducing expression of said recombinant product by introducing into said host cells one or more ligands for said modified ecdysone receptor, and optionally a silent partner for said modified ecdysone receptor.
22 more elements
Dependencies are inferred from the claim text.