Ecdysone receptor-based inducible gene expression system
View Patent ↗This invention relates to the field of biotechnology or genetic engineering. Specifically, this invention relates to the field of gene expression. More specifically, this invention relates to a novel inducible gene expression system and methods of modulating gene expression in a host cell for applications such as gene therapy, large scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic plants and animals.
1. A polynucleotide sequence encoding a gene switch comprising:
(a) a first gene expression cassette comprising a polynucleotide sequence that encodes a first polypeptide comprising:
(i) a DNA-binding domain that recognizes a response element associated with a gene of interest; and
(ii) an ecdysone receptor ligand binding domain;
(b) a second gene expression cassette comprising a polynucleotide sequence that encodes a second polypeptide comprising:
(i) a transactivation domain; and
(ii) a retinoid X receptor ligand binding domain;
wherein the transactivation domain is from a nuclear receptor other than an ecdysone receptor, a retinoid X receptor, or an ultraspiracle receptor,
wherein the first and second polypeptides dimerize
wherein at least one of the first polypeptide and the second polypeptide does not contain the A and B domains of the corresponding ecdysone receptor or retinoid X receptor and
wherein said gene switch is more sensitive to a diacylhydrazine ligand than to a steroid ligand when expressed in a mammalian cell.
2. The polynucleotide sequence according to claim 1 , further comprising a third gene expression cassette comprising:
(i) a response element to which the DNA-binding domain of the first polypeptide binds;
(ii) a promoter that is activated by the transactivation domain of the second polypeptide; and
(iii) the gene of interest.
3. The polynucleotide sequence according to claim 1 , wherein the ligand binding domain of the first polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.
4. The polynucleotide sequence according to claim 1 , wherein the ligand binding domain of the first polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.
5. The polynucleotide sequence according to claim 1 , wherein the ligand binding domain of the second polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.
6. The polynucleotide sequence according to claim 1 , wherein the ligand binding domain of the second polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40.
7. A polynucleotide sequence encoding a gene switch comprising:
(a) a first gene expression cassette comprising a polynucleotide sequence that encodes a first polypeptide comprising:
(i) a DNA-binding domain; and
(ii) a retinoid X receptor ligand binding domain; and
(b) a second gene expression cassette comprising a polynucleotide sequence that encodes a second polypeptide comprising:
(i) a transactivation domain; and
(ii) an ecdysone receptor ligand binding domain;
wherein the transactivation domain is from a nuclear receptor other than an ecdysone receptor, a retinoid X receptor, or an ultraspiracle receptor,
wherein the first and second polypeptides dimerize,
wherein at least one of the first polypeptide and the second polypeptide does not contain the A and B domains of the corresponding retinoid X receptor or ecdysone receptor and
wherein said gene switch is more sensitive to a diacylhydrazine ligand than to a steroid ligand when expressed in a mammalian cell.
8. The polynucleotide sequence according to claim 7 , further comprising a third gene expression cassette comprising:
(i) a response element to which the DNA-binding domain of the first polypeptide binds;
(ii) a promoter that is activated by the transactivation domain of the second polypeptide; and
(iii) a gene of interest.
9. The polynucleotide sequence according to claim 7 , wherein the ligand binding domain of the first polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40.
10. The polynucleotide sequence according to claim 7 , wherein the ligand binding domain of the second polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.
11. A vector comprising the polynucleotide sequence switch of claim 1 .
12. An isolated host cell comprising the polynucleotide sequence of claim 1 .
13. The isolated host cell according to claim 12 , wherein the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, a plant cell, and an animal cell.
14. The polynucleotide sequence of claim 1 , wherein said first polypeptide does not contain the ecdysone receptor A and B domains, and said second polypeptide does not contain the retinoid X receptor A and B domains.
15. The polynucleotide sequence of claim 7 , wherein said first polypeptide does not contain the retinoid X receptor A and B domains, and said second polypeptide does not contain the ecdysone receptor A and B domains.
16. The polynucleotide sequence of claim 1 , wherein said diacylhydrazine ligand is a compound of the formula
wherein:
E is a (C4-C6)alkyl containing a tertiary carbon or a cyano(C 3 -C 5 ) alkyl containing a tertiary carbon;
R 1 is H, Me, Et, i-Pr, F, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, SCN, or SCHF 2 ;
R 2 is H, Me, Et, n-Pr, i-Pr, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe 2 , NEt 2 , SMe, SEt, SOCF 3 , OCF 2 CF 2 H, COEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, OCF 3 , OCHF 2 , O-i-Pr, SCN, SCHF 2 , SOMe, NH—CN, or joined with R 3 and the phenyl carbons to which R 2 and R 3 are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;
R 3 is H, Et, or joined with R 2 and the phenyl carbons to which R 2 and R 3 are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;
R 4 , R 5 , and R 6 are independently H, Me, Et, F, Cl, Br, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt.
17. The polynucleotide sequence of claim 7 , wherein said diacylhydrazine ligand is a compound of the formula
wherein:
E is a (C4-C6)alkyl containing a tertiary carbon or a cyano(C 3 -C 5 ) alkyl containing a tertiary carbon;
R 1 is H, Me, Et, i-Pr, F, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, SCN, or SCHF 2 ;
R 2 is H, Me, Et, n-Pr, i-Pr, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe 2 , NEt 2 , SMe, SEt, SOCF 3 , OCF 2 CF 2 H, COEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, OCF 3 , OCHF 2 , O-i-Pr, SCN, SCHF 2 , SOMe, NH—CN, or joined with R 3 and the phenyl carbons to which R 2 and R 3 are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;
R 3 is H, Et, or joined with R 2 and the phenyl carbons to which R 2 and R 3 are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;
R 4 , R 5 , and R 6 are independently H, Me, Et, F, Cl, Br, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt.
18. The polynucleotide sequence of claim 1 , wherein said polynucleotide sequence further comprises a tissue-specific promoter.
19. The polynucleotide sequence of claim 7 , wherein said polynucleotide sequence further comprises a tissue-specific promoter.
20. A vector comprising the polynucleotide sequence switch of claim 7 .
21. An isolated host cell comprising the polynucleotide sequence claim 7 .
22. The isolated host cell according to claim 21 , wherein the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, a plant cell, and an animal cell.