Mutant Receptors and Their Use in a Nuclear Receptor-Based Inducible Gene Expression

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 novel substitution mutant receptors and their use in a nuclear receptor-based inducible gene expression system and methods of modulating the expression of a gene 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 organisms.

1 - 21 . (canceled) 
     
     
         22 . A polynucleotide comprising a sequence encoding a polypeptide comprising a DNA-binding domain and a ecdysone receptor ligand binding domain, wherein the ecdysone receptor ligand binding domain comprises a substitution mutation at a position or positions corresponding to:
 a) amino acid residue 132 of SEQ ID NO: 1,   b) both of amino acid residues 96 and 119 of SEQ ID NO: 1,   c) both of amino acid residues 110 and 128 of SEQ ID NO: 1,   d) both of amino acid residues 52 and 110 of SEQ ID NO: 1,   e) both of amino acid residues 125 and 132 of SEQ ID NO: 1,   f) all three of amino acid residues 107, 110, and 127 of SEQ ID NO: 1,   g) all three of amino acid residues 52, 107 and 127 of SEQ ID NO: 1,   h) all three of amino acid residues 107, 127 and 259 of SEQ ID NO: 1, or   i) an amino acid substitution corresponding to at least one of F48Y, F48W, F48L, F48N, F48R, F48K, I51M, I51N, I51L, T52M, T52E, T52P, T52R, T52W, T52G, T52Q, M54W, M54T, M92L, M92E, R95H, R95M, R95W, V96L, V96W, V96S, V96E, F109W, F109P, F109L, 109M, F109N, A110E, A110N, A110W, N119F, Y120W, Y120M, M125P, M125R, M125E, M125L, M125C, M125W, M125G, M125I, M125N, M125S, M125V, V128F, M219K, M219W, M219Y, M219A, L223K, L223R, L223Y, L234M, L234I, L234R, L234W, W238P, W238E, W238Y, W238M, and W238L.   
     
     
         23 . The polynucleotide of  claim 22 , wherein the ecdysone receptor ligand binding domain is from a spruce budworm  Choristoneura fumiferana  ecdysone receptor. 
     
     
         24 . The polynucleotide of  claim 23 , wherein the DNA-binding domain is a GAL4 DNA-binding domain. 
     
     
         25 . An expression vector comprising the polynucleotide of  claim 22 . 
     
     
         26 . An expression vector comprising the polynucleotide of  claim 23 . 
     
     
         27 . An expression vector comprising the polynucleotide of  claim 24 . 
     
     
         28 . An isolated host cell comprising the polynucleotide of  claim 22 . 
     
     
         29 . An isolated host cell comprising the polynucleotide of  claim 23 . 
     
     
         30 . An isolated host cell comprising the polynucleotide of  claim 24 . 
     
     
         31 . An isolated host cell comprising the expression vector of  claim 25 . 
     
     
         32 . The isolated host cell of  claim 28 , wherein the host cell is a cell selected from the group consisting of:
 a) a bacterial cell;   b) a fungal cell;   c) a nematode cell;   d) an insect cell;   e) a fish cell;   f) a plant cell;   g) an avian cell;   h) an animal cell; and   i) a mammalian cell.   
     
     
         33 . A non-human organism transformed with the polynucleotide of  claim 22 . 
     
     
         34 . A non-human organism transformed with the polynucleotide of  claim 23 . 
     
     
         35 . A non-human organism transformed with the polynucleotide of  claim 24 . 
     
     
         36 . A polypeptide comprising a DNA-binding domain and a ecdysone receptor ligand binding domain, wherein the ecdysone receptor ligand binding domain comprises a substitution mutation at a position or positions corresponding to:
 a) amino acid residue 132 of SEQ ID NO: 1,   b) both of amino acid residues 96 and 119 of SEQ ID NO: 1,   c) both of amino acid residues 110 and 128 of SEQ ID NO: 1,   d) both of amino acid residues 52 and 110 of SEQ ID NO: 1,   e) both of amino acid residues 125 and 132 of SEQ ID NO: 1,   f) all three of amino acid residues 107, 110, and 127 of SEQ ID NO: 1,   g) all three of amino acid residues 52, 107 and 127 of SEQ ID NO: 1,   h) all three of amino acid residues 107, 127 and 259 of SEQ ID NO: 1, or   i) an amino acid substitution corresponding to at least one of F48Y, F48W, F48L, F48N, F48R, F48K, I51M, 151N, I51L, T52M, T52E, T52P, T52R, T52W, T52G, T52Q, M54W, M54T, M92L, M92E, R95H, R95M, R95W, V96L, V96W, V96S, V96E, F109W, F109P, F109L, F109M, F109N, A110E, A110N, A110W, N119F, Y120W, Y120M, M125F, M125R, M125E, M125L, M125C, M125W, M125G, M125I, M125N, M125S, M125V, V128F, M219K, M219W, M219Y, M219A, L223K, L223R, L223Y, L234M, L234I , L234R, L234W, W238P, W238E, W238Y, W238M, and W238L.   
     
     
         37 . The polypeptide of  claim 36 , wherein the ecdysone receptor ligand binding domain is from a spruce budworm  Choristoneura fumiferana  ecdysone receptor. 
     
     
         38 . The polypeptide of  claim 36 , wherein the DNA-binding domain is a GAL4 DNA-binding domain.