US2004033600A1PendingUtilityA1

Ecdysone receptor-based inducible gene expression system

56
Priority: Mar 21, 2001Filed: Mar 21, 2001Published: Feb 19, 2004
Est. expiryMar 21, 2021(expired)· nominal 20-yr term from priority
Inventors:Subba Reddy PalliMarianna Zinovjevna KapitskayaDean Ervin Cress
C12N 2830/15C12N 15/1055C12N 15/63C12N 2830/002C12N 2510/00C07K 2319/80C07K 2319/71C12N 15/85C07K 2319/715
56
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Claims

Abstract

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.

Claims

exact text as granted — not AI-modified
We claim:  
     
         1 . A gene expression modulation system comprising: 
 a) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide sequence that encodes a first polypeptide comprising: 
 i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated;  
 i) a ligand binding domain comprising a ligand binding domain from a nuclear receptor;  
   b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide sequence that encodes a second polypeptide comprising: 
 i) a transactivation domain; and  
 ii) a ligand binding domain comprising a ligand binding domain from a nuclear receptor other than ultraspiracle (USP);  
 wherein the transactivation domain is from a nuclear receptor other than an ecdysone receptor, a retinoid X receptor, or an ultraspiracle receptor; and wherein the ligand binding domains from the first polypeptide and the second polypeptide are different and dimerize.  
   
     
     
         2 . The gene expression modulation system 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 whose expression is to be modulated.    
     
     
         3 . The gene expression modulation system according to  claim 1 , wherein the ligand binding domain of the first polypeptide is an ecdysone receptor polypeptide.  
     
     
         4 . The gene expression modulation system according to  claim 1 , wherein the ligand binding domain of the second polypeptide is a retinoid X receptor polypeptide.  
     
     
         5 . A gene expression modulation system comprising: 
 a) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide sequence that encodes a first polypeptide comprising: 
 i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and  
 ii) a ligand binding domain comprising a ligand binding domain from an ecdysone receptor; and  
   b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide sequence that encodes a second polypeptide comprising: 
 i) a transactivation domain; and  
 ii) a ligand binding domain comprising a ligand binding domain from a retinoid X receptor;  
 wherein the ligand binding domains from the first polypeptide and the second polypeptide are different and dimerize.  
   
     
     
         6 . The gene expression modulation system according to  claim 5 , further comprising a third gene expression cassette comprising: 
 i) a response element to which the DNA-binding domain of the fust polypeptide binds;    ii) a promoter that is activated by the transactivation domain of the second polypeptide; and    iii) the gene whose expression is to be modulated.    
     
     
         7 . The gene expression modulation system according to  claim 5 , 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 D NO: 10.  
     
     
         8 . The gene expression modulation system according to  claim 5 , 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.  
     
     
         9 . The gene expression modulation system according to  claim 5 , 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.  
     
     
         10 . The gene expression modulation system according to  claim 5 , 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 NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.  
     
     
         11 . A gene expression modulation system comprising: 
 a) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide sequence that encodes a first polypeptide comprising: 
 i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and  
 ii) a ligand binding domain comprising a ligand binding domain from a retinoid X receptor; and  
   b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide sequence that encodes a second polypeptide comprising: 
 i) a transactivation domain; and  
 ii) a ligand binding domain comprising a ligand binding domain from an ecdysone receptor;  
 wherein the ligand binding domains from the first polypeptide and the second polypeptide are different and dimerize.  
   
     
     
         12 . The gene expression modulation system according to  claim 11 , 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 whose expression is to be modulated.    
     
     
         13 . The gene expression modulation system according to  claim 11 , 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: 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.  
     
     
         14 . The gene expression modulation system according to  claim 11 , 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.  
     
     
         15 . The gene expression modulation system according to  claim 11 , 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: 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.  
     
     
         16 . The gene expression modulation system according to  claim 11 , 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.  
     
     
         17 . A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a DNA-binding domain and an ecdysone receptor ligand binding domain, wherein the DNA binding domain is from a nuclear receptor other than an ecdysone receptor.  
     
     
         18 . The gene expression cassette according to  claim 18 , wherein the DNA-binding domain is a GAL4 DNA-binding domain or a LexA DNA-binding domain.  
     
     
         19 . A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a DNA-binding domain and a retinoid X receptor ligand binding domain, wherein the DNA binding domain is from a nuclear receptor other than a retinoid X receptor.  
     
     
         20 . The gene expression cassette according to  claim 19 , wherein the DNA-binding domain is a GALA DNA-binding domain or a LexA DNA-binding domain.  
     
     
         21 . A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a transactivation domain and an ecdysone receptor ligand binding domain, wherein the transactivation domain is from a nuclear receptor other than an ecdysone receptor.  
     
     
         22 . The gene expression cassette according to  claim 21 , wherein the transactivation domain is a VP16 transactivation domain.  
     
     
         23 . A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a transactivation domain and a retinoid X receptor ligand binding domain, wherein the transactivation domain is from a nuclear receptor other than a retinoid X receptor.  
     
     
         24 . The gene expression cassette according to  claim 22 , wherein the transactivation domain is a VP16 transactivation domain.  
     
     
         25 . A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a DNA-binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 41) or a LexA DBD (SEQ ID NO: 43) and an ecdysone receptor ligand binding domain 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.  
     
     
         26 . A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a DNA-binding domain comprising an amino acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 42) or a LexA DBD (SEQ ID NO: 44) and an ecdysone receptor ligand binding domain comprising 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.  
     
     
         27 . A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a DNA-binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of a GALA DBD (SEQ ID NO: 41) or a LexA DBD (SEQ ID NO: 43) and a retinoid X receptor ligand binding domain 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.  
     
     
         28 . A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a DNA-binding domain comprising an amino acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 42) or a LexA DBD (SEQ ID NO: 44) and a retinoid X receptor ligand binding domain comprising 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.  
     
     
         29 . A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a transactivation domain encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 45 and an ecdysone receptor ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting ofSEQ 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.  
     
     
         30 . A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a transactivation domain comprising an amino acid sequence of SEQ ID NO: 46 and an ecdysone receptor ligand binding domain comprising 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.  
     
     
         31 . A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a transactivation domain encoded by a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 45 and a retinoid X receptor ligand binding domain 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.  
     
     
         32 . A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a transactivation domain comprising an amino acid sequence of SEQ ID NO: 46 and a retinoid X receptor ligand binding domain comprising 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.  
     
     
         33 . An isolated polynucleotide encoding an ecdysone receptor polypeptide or a retinoid X receptor polypeptide comprising a truncation mutation, wherein the truncation mutation reduces ligand binding activity of the ecdysone receptor polypeptide or the retinoid X receptor polypeptide.  
     
     
         34 . An isolated polynucleotide encoding an ecdysone receptor polypeptide or a retinoid X receptor polypeptide comprising a truncation mutation, wherein the truncation mutation reduces steroid binding activity of the ecdysone receptor polypeptide or the retinoid X receptor polypeptide.  
     
     
         35 . An isolated polynucleotide encoding an ecdysone receptor polypeptide or a retinoid X receptor polypeptide comprising a truncation mutation, wherein the truncation mutation reduces non-steroid binding activity of the ecdysone receptor polypeptide or the retinoid X receptor polypeptide.  
     
     
         36 . An isolated polynucleotide encoding an ecdysone receptor polypeptide or a retinoid X receptor polypeptide comprising a truncation mutation, wherein the truncation mutation enhances ligand binding activity of the ecdysone receptor polypeptide or the retinoid X receptor polypeptide.  
     
     
         37 . An isolated polynucleotide encoding an ecdysone receptor polypeptide or a retinoid X receptor polypeptide comprising a truncation mutation, wherein the truncation mutation enhances steroid binding activity of the ecdysone receptor polypeptide or the retinoid X receptor polypeptide.  
     
     
         38 . An isolated polynucleotide encoding an ecdysone receptor polypeptide or a retinoid X receptor polypeptide comprising a truncation mutation, wherein the truncation mutation enhances non-steroid binding activity of the ecdysone receptor polypeptide or the retinoid X receptor polypeptide.  
     
     
         39 . An isolated polynucleotide encoding a retinoid X receptor polypeptide comprising a truncation mutation, wherein the truncation mutation increases ligand sensitivity of the retinoid X receptor polypeptide.  
     
     
         40 . An isolated polynucleotide encoding a retinoid X receptor polypeptide comprising a truncation mutation, wherein the truncation mutation increases ligand sensitivity of a heterodimer, wherein the heterodimer comprises said retinoid X receptor polypeptide and a dimerization partner.  
     
     
         41 . The isolated polynucleotide according to  claim 40 , wherein the dimerization partner is an ecdysone receptor polypeptide.  
     
     
         42 . An isolated polynucleotide encoding a truncated ecdysone receptor polypeptide, wherein the polynucleotide comprises 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.  
     
     
         43 . An isolated polypeptide encoded by the isolated polynucleotide according to  claim 42 .  
     
     
         44 . An isolated truncated ecdysone receptor polypeptide comprising 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.  
     
     
         45 . An isolated polynucleotide encoding a truncated retinoid X receptor polypeptide, wherein the polynucleotide comprises 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.  
     
     
         46 . An isolated polypeptide encoded by the isolated polynucleotide according to  claim 45 .  
     
     
         47 . An isolated truncated retinoid X receptor polypeptide comprising 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.  
     
     
         48 . A method of modulating the expression of a gene in a host cell comprising the gene to be modulated comprising the steps of: 
 a) introducing into the host cell the gene expression modulation system according to  claim 1;  and    b) introducing into the host cell a ligand that independently combines with the ligand binding domains of the first polypeptide and the second polypeptide;    wherein the gene to be expressed is a component of a chimeric gene comprising:    i) a response element to which the DNA binding domain from the first polypeptide binds;    ii) a promoter that is activated by the transactivation domain of the second polypeptide; and    iii) a gene whose expression is to be modulated,    whereby a complex is formed comprising the ligand, the first polypeptide, and the second polypeptide, and whereby the complex modulates expression of the gene in the host cell.    
     
     
         49 . The method according to  claim 48 , wherein the ligand is a compound of the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 E is a (C 4 -C 6 )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.  
 
     
     
         50 . A method of modulating the expression of a gene in a host cell comprising the gene to be modulated comprising the steps of: 
 a) introducing into the host cell the gene expression modulation system of  claim 5;  and    b) introducing into the host cell a ligand that independently combines with the ligand binding domains of the first polypeptide and the second polypeptide;    wherein the gene to be expressed is a component of a chimeric gene comprising:    i) a response element to which the DNA binding domain from the first polypeptide binds;    ii) a promoter that is activated by the transactivation domain of the second polypeptide; and    iii) a gene whose expression is to be modulated,    whereby a complex is formed comprising the ligand, the first polypeptide, and the second polypeptide, and whereby the complex modulates expression of the gene in the host cell.    
     
     
         51 . The method according to  claim 50 , wherein the ligand is a compound of the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 E is a (C 4 -C)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 7 , 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-nPr, OAc, NMe2, 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 dilydropyryl 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.  
 
     
     
         52 . A method of modulating the expression of a gene in a host cell comprising the gene to be modulated comprising the steps of: 
 a) introducing into the host cell the gene expression modulation system of  claim 11;  and    b) introducing into the host cell a ligand that independently conibines with the ligand binding domains of the first polypeptide and the second polypeptide;    wherein the gene to be expressed is a component of a chimeric gene comprising:    i) a response element to which the DNA binding domain from the first polypeptide binds;    ii) a promoter that is activated by the transactivation domain of the second polypeptide; and    iii) a gene whose expression is to be modulated,    whereby a complex is formed comprising the ligand, the first polypeptide, and the second polypeptide, and whereby the complex modulates expression of the gene in the host cell.    
     
     
         53 . The method according to  claim 52 , wherein the ligand is a compound of the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 E is a (C 4 -C 6 )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-nPr, OAc, NMe 2 , NEt 2 , SMe, SEt, SOCF 3 , OCF 2 CF 2 H, COEt, cyclopropyl, CF2CF3, 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.  
 
     
     
         54 . An isolated host cell into which the gene expression modulation system according to  claim 1  has been introduced.  
     
     
         55 . The isolated host cell according to  claim 54 , wherein the host cell is selected from the group consisting of abacterial cell, a fungal cell, a yeast cell, a plant cell, an animal cell, and a mammalian cell.  
     
     
         56 . The isolated host cell according to  claim 55 , wherein the host cell is a plant cell, a murine cell, or a human cell.  
     
     
         57 . An isolated host cell into which the gene expression modulation system according to  claim 5  has been introduced.  
     
     
         58 . The isolated host cell according to  claim 57 , wherein the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, a plant cell, an animal cell, and a mammalian cell.  
     
     
         59 . The isolated host cell according to  claim 58 , wherein the host cell is a plant cell, a murine cell, or a human cell.  
     
     
         60 . An isolated host cell into which the gene expression modulation system according to  claim 11  has been introduced.  
     
     
         61 . The isolated host cell according to  claim 60 , wherein the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, a plant cell, an animal cell, and a mammalian cell.  
     
     
         62 . The isolated host cell according to  claim 61 , wherein the host cell is a plant cell, a murine cell, or a human cell.  
     
     
         63 . A non-human organism comprising a host cell into which the gene expression modulation system according to  claim 1  has been introduced.  
     
     
         64 . The non-human organism according to  claim 63 , wherein the non-human organism is selected from the group consisting of a bacterium, a fungus, a yeast, a plant, an animal, and a mammal.  
     
     
         65 . The non-human organism according to  claim 64 , wherein the non-human organism is selected from the group consisting of a plant, a mouse, a rat, a rabbit, a cat, a dog, a bovine, a goat, a pig, a horse, a sheep, a monkey, and a chimpanzee.  
     
     
         66 . A non-human organism comprising a host cell into which the gene expression modulation system according to claim S has been introduced.  
     
     
         67 . The non-human organism according to  claim 66 , wherein the non-human organism is selected from the group consisting of a bacterium, a fungus, a yeast, a plant, an animal, and a mammal.  
     
     
         68 . The non-human organism according to  claim 67 , wherein the non-human organism is selected from the group consisting of a plant, a mouse, a rat, a rabbit, a cat, a dog, a bovine, a goat, a pig, a horse, a sheep, a monkey, and a chimpanzee.  
     
     
         69 . A non-human organism comprising a host cell into which the gene expression modulation system according to  claim 11  has been introduced.  
     
     
         70 . The non-human organism according to  claim 69 , wherein the non-human organism is selected from the group consisting of a bacterium, a fungus, a yeast, a plant, an animal, and a mammal.  
     
     
         71 . The non-human organism according to  claim 70 , wherein the non-human organism is selected from the group consisting of a plant, a mouse, a rat, a rabbit, a cat, a dog, a bovine, a goat, a pig, a horse, a sheep, a monkey, and a chimpanzee.

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