Multiple Inducible Gene Regulation System

Abstract

The present invention relates to the field of biotechnology or genetic engineering. More specifically, the present invention relates to a multiple inducible gene regulation system that functions within cells to simultaneously control the quantitative expression of multiple genes.

1 .- 20 . (canceled) 
     
     
         21 . A non-human organism comprising a multiple inducible gene modulation system comprising two individually operable heterologous gene modulation systems,
 wherein each individually operable heterologous gene modulation system comprises:   a) a first gene expression cassette comprising a polynucleotide sequence that encodes a polypeptide comprising:
 A) a DNA binding domain that recognizes a response element associated with a gene of interest; 
 B) an ecdysone receptor ligand binding domain; and 
   b) a second gene expression cassette comprising a polynucleotide sequence comprising a promoter operably linked to a polynucleotide sequence that encodes a second polypeptide comprising a nuclear receptor ligand binding domain and a transactivation domain; and   wherein each individually operable heterologous gene modulation system is orthogonal to the other individually operable heterologous gene modulation system present in the multiple inducible gene modulation system.   
     
     
         22 . The non-human organism of  claim 21 , wherein said organism is a transgenic organism. 
     
     
         23 . The non-human organism of  claim 21 , wherein said organism is an isolated cell. 
     
     
         24 . The non-human organism of  claim 21 , wherein said nuclear receptor ligand binding domain is selected from the group consisting of a vertebrate retinoid X receptor ligand binding domain; an invertebrate retinoid X receptor ligand binding domain; an ultraspiracle protein ligand binding domain; and a chimeric ligand binding domain comprising two polypeptide fragments, wherein the first polypeptide fragment is from a vertebrate retinoid X receptor ligand binding domain, an invertebrate retinoid X receptor ligand binding domain, or an ultraspiracle protein ligand binding domain, and the second polypeptide fragment is from a different vertebrate retinoid X receptor ligand binding domain, invertebrate retinoid X receptor ligand binding domain, or an ultraspiracle protein ligand binding domain. 
     
     
         25 . The non-human organism of  claim 21 , wherein said 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, an Orthopteran ecdysone receptor ligand binding domain, a Homopteran ecdysone receptor ligand binding domain, a Hemipteran ecdysone receptor ligand binding domain, a spruce budworm  Choristoneura fumiferana  ecdysone receptor ligand binding domain, a yellow meal worm  Tenebrio molitor  ecdysone receptor ligand binding domain, a tobacco hornworm  Manduca sexta  ecdysone receptor ligand binding domain, a tobacco budworm  Heliothies virescens  ecdysone receptor ligand binding domain, a golmidge  Chironomus tentans  ecdysone receptor ligand binding domain, a silkworm  Bombyx mori  ecdysone receptor ligand binding domain, a squinting bush brown  Bicyclus anynana  ecdysone receptor ligand binding domain, a buckeye  Junonia coenia  ecdysone receptor ligand binding domain, a fruit fly  Drosophila melanogaster  ecdysone receptor ligand binding domain, a yellow fever mosquito  Aedes aegypti  ecdysone receptor ligand binding domain, a blowfly  Lucilia capitata  ecdysone receptor ligand binding domain, a sheep blowfly  Lucilia cuprina  ecdysone receptor ligand binding domain, a blowfly  Calliphora vicinia  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  Celuca pugilator  ecdysone receptor ligand binding domain, an ixodid tick  Amblyomma americanum  ecdysone receptor ligand binding domain, and a white fly  Bamecia argentifoli  ecdysone receptor ligand binding domain. 
     
     
         26 . The non-human organism of  claim 25 , wherein said ecdysone receptor ligand binding domain is a spruce budworm  Choristoneura fumiferana  ecdysone receptor ligand binding domain. 
     
     
         27 . The non-human organism of  claim 21 , wherein said 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 Group H nuclear receptor member DNA binding domain, a steroid/thyroid hormone nuclear receptor superfamily member DNA binding domain, a bacterial LacZ DNA binding domain, and an ecdysone receptor DNA binding domain. 
     
     
         28 . The non-human organism of  claim 21 , wherein said transactivation domain is selected from the group consisting of a Group H nuclear receptor member transactivation domain, 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-KB transactivation domain, a BP64 transactivation domain, a B42 acidic transactivation domain, and a p65 transactivation domain. 
     
     
         29 . The non-human organism of  claim 21 , wherein said organism is a prokaryote. 
     
     
         30 . The non-human organism of  claim 21 , wherein said organism is an invertebrate. 
     
     
         31 . The non-human organism of  claim 21  wherein said organism is selected from the group consisting of a bacterium, a fungus and a yeast. 
     
     
         32 . The non-human organism of  claim 31 , wherein said bacterium is selected from the group consisting of  Synechocystis, Synechococcus, Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces, Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcaligenes, Synechocystis, Anabaena, Thiobacillus, Methanobacterium  and  Klebsiella.    
     
     
         33 . The non-human organism of  claim 31 , wherein said yeast is selected from the group consisting of  Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida  and  Hansenula.    
     
     
         34 . The non-human organism of  claim 21 , wherein said organism is an animal. 
     
     
         35 . The non-human organism of  claim 34 , wherein said animal is a mammal. 
     
     
         36 . The non-human organism of  claim 35 , wherein said mammal is selected from the group consisting of a hamster, a mouse, a rat, a rabbit, a cat, a dog, a bovine, a goat, a pig, a horse and a sheep. 
     
     
         37 . The non-human organism of  claim 34 , wherein said animal is an avian. 
     
     
         38 . The non-human organism of  claim 37 , wherein said avian is a chicken. 
     
     
         39 . The non-human organism of  claim 21 , wherein said organism is a plant. 
     
     
         40 . The non-human organism of  claim 39 , wherein said plant is selected from the group consisting of an apple, an  Arabidopsis , a bajra, a banana, a barley, a bean, a beet, a blackgram, a chickpea, a chili, a cucumber, an eggplant, a favabean, a maize, a melon, a millet, a mungbean, an oat, an okra, a  Panicum , a papaya, a peanut, a pea, a pepper, a pigeonpea, a pineapple, a  Phaseolus , a potato, a pumpkin, a rice, a sorghum, a soybean, a squash, a sugarcane, a sugarbeet, a sunflower, a sweet a potato, a tea, a tomato, a tobacco, a watermelon, and a wheat. 
     
     
         41 . The non-human organism of  claim 21 , wherein each individually operable heterologous gene modulation system is associated with an expression cassette comprising a response element to which the DNA-binding domain binds, a promoter that is activated by the transactivation domain, and a gene of interest.