Novel Ecdysone Receptor-Based Inducible Gene Expression System

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.

1 - 71 . (canceled) 
     
     
         72 . A method of modulating the expression of a gene in a host cell comprising the gene to be modulated, the method comprising:
 (a) introducing into the host cell a gene expression modulation system comprising
 (i) a first gene expression cassette comprising a polynucleotide sequence that encodes a first polypeptide comprising an ecdysone receptor ligand binding domain; and 
 (ii) a second gene expression cassette comprising a polynucleotide sequence that encodes a second polypeptide comprising a nuclear receptor ligand binding domain that is not an ultraspiracle ligand binding domain; and 
   (b) introducing into the host cell a ligand that binds to the Group H nuclear receptor ligand binding domain,   wherein one of the first gene expression cassette or the second gene expression cassette comprises a DNA-binding domain that recognizes a response element associated with a gene of interest,   wherein the first gene expression cassette or the second gene expression cassette that does not comprise the DNA-binding domain comprises a transactivation domain that is not an ecdysone receptor transactivation domain, a retinoid X receptor transactivation domain, or an ultraspiracle receptor transactivation domain,   wherein the ligand binding domain in the first polypeptide and the ligand binding domain in the second polypeptide are different and dimerize, and   wherein the gene that is expressed is a component of a chimeric gene comprising:   (i) a response element to which the DNA-binding binds;   (ii) a promoter that is activated by the transactivation domain; and   (iii) the gene that is expressed.   
     
     
         73 . The method of  claim 72 , wherein the gene expression modulation system further comprises:
 (iii) a third gene expression cassette comprising
 (A) a response element to which the DNA-binding domain of the first polypeptide binds; 
 (B) a promoter that is activated by the transactivation domain of the second polypeptide; and 
 (C) the gene that is expressed. 
   
     
     
         74 . The method of  claim 72 , wherein the ligand is a diacylhydrazine. 
     
     
         75 . The method of  claim 74 , 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. 
       
     
     
         76 . The method of  claim 72 , 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. 
     
     
         77 . The method of  claim 72 , 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. 
     
     
         78 . The method of  claim 72 , 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. 
     
     
         79 . The method of  claim 72 , 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. 
     
     
         80 . The method of  claim 72 , wherein in the gene expression modulation system is contained in a vector. 
     
     
         81 . The method of  claim 80 , wherein the vector is a plasmid. 
     
     
         82 . The method of  claim 80 , wherein the vector is an expression vector. 
     
     
         83 . The method of  claim 80 , wherein the vector is a viral vector. 
     
     
         84 . The method of  claim 83 , wherein the viral vector is an adenovirus vector. 
     
     
         85 . The method of  claim 72 , wherein the ecdysone receptor ligand binding domain is selected from the group consisting of a  Lepidopteran  ecdysone receptor ligand binding domain, a  Dipteran  ecdysone receptor ligand binding domain, an  Arthropod  ecdysone receptor ligand binding domain, a  Homopteran  ecdysone receptor ligand binding domain, a spruce budworm  Choristoneura fumiferana  ecdysone receptor ligand binding domain, a  Tenebrio molitor  ecdysone receptor ligand binding domain, a  Manduca sexta  ecdysone receptor ligand binding domain, a  Heliothies virescens  ecdysone receptor ligand binding domain, a silk moth  Bombyx mori  ecdysone receptor ligand binding domain, a fruit fly  Drosophila melanogaster  ecdysone receptor ligand binding domain, a mosquito  Aedes aegypti  ecdysone receptor ligand binding domain, a blowfly  Lucilia capitata  ecdysone receptor ligand binding domain, a Mediterranean fruit fly  Ceratitis capitata  ecdysone receptor ligand binding domain, a locust  Locusta migratoria  ecdysone receptor ligand binding domain, an aphid  Myzus persicae  ecdysone receptor ligand binding domain, a fiddler crab  Uca pugilator  ecdysone receptor ligand binding domain, and an ixodid tick  Amblyomma americanum  ecdysone receptor ligand binding domain. 
     
     
         86 . The method of  claim 85 , wherein the ecdysone receptor is  Choristoneura fumiferana  ecdysone receptor ligand binding domain. 
     
     
         87 . The method of  claim 72 , wherein the expression of the gene is tissue-specific expression. 
     
     
         88 . The method of  claim 72 , wherein the first polypeptide does not contain the A and B domains of the ecdysone receptor. 
     
     
         89 . The method of  claim 72 , wherein the second polypeptide does not contain the A and B domains of the nuclear receptor. 
     
     
         90 . The method of  claim 72 , wherein the first polypeptide does not contain the A and B domains of the Group H nuclear receptor, and
 wherein the second polypeptide does not contain the A and B domains of the nuclear receptor.   
     
     
         91 . The method of  claim 72 , wherein the gene expression modulation system is more sensitive to a diacylhydrazine ligand than to a steroid ligand. 
     
     
         92 . The method of  claim 91 , wherein the gene expression modulation system is more sensitive to a diacylhydrazine ligand than to a steroid ligand when expressed in a mammalian cell. 
     
     
         93 . The method of  claim 72 , wherein the DNA binding domain is selected from the group consisting of a GAL4 DNA binding domain, a LexA DNA binding domain, a transcription factor DNA binding domain, a steroid/thyroid hormone nuclear receptor superfamily member DNA binding domain and a bacterial LacZ DNA binding domain. 
     
     
         94 . The method of  claim 72 , wherein the transactivation domain is selected from the group consisting of a steroid/thyroid hormone nuclear receptor transactivation domain, a polyglutamine transactivation domain, a basic or acidic amino acid transactivation domain, a VP16 transactivation domain, a GAL4 transactivation domain, an NF-κB transactivation domain and a BP64 transactivation domain. 
     
     
         95 . The method of  claim 72 , wherein the nuclear receptor ligand binding domain of the second polypeptide is a retinoic X receptor ligand binding domain. 
     
     
         96 . The method of  claim 95 , wherein the retinoic X receptor ligand binding domain of the second polypeptide is selected from the group consisting of a mouse  Mus musculus  retinoic X receptor ligand binding domain, a human  Homo sapiens  retinoic X receptor ligand binding domain. 
     
     
         97 . The method of  claim 95 , wherein the retinoic X receptor ligand binding domain of the second polypeptide is selected from the group consisting of an RXRα ligand binding domain, an RXRβ ligand binding domain and an RXRγ ligand binding domain. 
     
     
         98 . The method of  claim 72 , 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, a mammalian cell, a mouse cell, and a human cell. 
     
     
         99 . The method of  claim 98 , wherein the host cell is selected from the group consisting of an  Aspergillus  cell, a  Trichoderma  cell, a  Saccharomyces  cell, a  Pichia  cell, a  Candida  cell, and a  Hansenula  cell. 
     
     
         100 . The method of  claim 98 , wherein the host cell is selected from the group consisting of a  Synechocystis  cell, a  Synechococcus  cell, a  Salmonella  cell, a  Bacillus  cell, an  Acinetobacter  cell, a  Rhodococcus  cell, a  Streptomyces  cell, an  Escherichia  cell, a  Pseudomonas  cell, a  Methylomonas  cell, a  Methylobacter  cell, an  Alcaligenes  cell, a  Synechocystis  cell, an  Anabaena  cell, a  Thiobacillus  cell, a  Methanobacterium  cell and a  Klebsiella  cell. 
     
     
         101 . The method of  claim 98 , wherein the host cell is a plant cell. 
     
     
         102 . The method of  claim 101 , wherein the plant cell is selected from the group consisting of an apple cell, an  Arabidopsis  cell, a bajra cell, a banana cell, a barley cell, a bean cell, a beet cell, a blackgram cell, a chickpea cell, a chili cell, a cucumber cell, an eggplant cell, a favabean cell, a maize cell, a melon cell, a millet cell, a mungbean cell, an oat cell, an okra cell, a  Panicum  cell, a papaya cell, a peanut cell, a pea cell, a pepper cell, a pigeonpea cell, a pineapple cell, a  Phaseolus  cell, a potato cell, a pumpkin cell, a rice cell, a sorghum cell, a soybean cell, a squash cell, a sugarcane cell, a sugarbeet cell, a sunflower cell, a sweet potato cell, a tea cell, a tomato cell, a tobacco cell, a watermelon cell, and a wheat cell. 
     
     
         103 . The method of  claim 98 , wherein host cell is a mammalian cell. 
     
     
         104 . The method of  claim 103 , wherein the mammalian cell is selected from the group consisting of a hamster cell, a mouse cell, a rat cell, a rabbit cell, a cat cell, a dog cell, a bovine cell, a goat cell, a cow cell, a pig cell, a horse cell, a sheep cell, a monkey cell, a chimpanzee cell, and a human cell. 
     
     
         105 . The method of  claim 98 , wherein the mammalian cell is a human cell. 
     
     
         106 . The method of  claim 72 , wherein the first polypeptide comprises a DNA binding domain and the second polypeptide comprises a transactivation domain. 
     
     
         107 . The method of  claim 72 , wherein the first polypeptide comprises a transactivation domain and the second polypeptide comprises a DNA binding domain.