Chimeric Retinoid X Receptors and Their Use in a Novel Ecdysone Receptor-Based Inducible Gene Expression System

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 hybrid polypeptide comprising:

i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and

ii) an ecdysone receptor ligand binding domain; 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 hybrid polypeptide comprising:

i) a transactivation domain; and

ii) a chimeric retinoid X receptor ligand binding domain.

2 . (canceled)

3 . The gene expression modulation system according to claim 1 , wherein the ecdysone receptor ligand binding domain of the first hybrid polypeptide is selected from the group consisting of a spruce budworm Choristoneura fumiferana ecdysone receptor ligand binding domain, a beetle Tenebrio monitor ecdysone receptor ligand binding domain, a Manduca sexta ecdysone receptor ligand binding domain, a Heliothies virescens ecdysone receptor ligand binding domain LBD, a midge Chironomus tentans ecdysone receptor ligand binding domain LBD, 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 blowfly Lucilia cuprina 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, a whitefly Bamecia argentifoli ecdysone receptor ligand binding domain, a leafhopper Nephotetix cincticeps ecdysone receptor ligand binding domain, and an ixodid tick Amblyomma americanum ecdysone receptor ligand binding domain.

4 - 5 . (canceled)

6 . The gene expression modulation system according to claim 1 , wherein the chimeric retinoid X receptor ligand binding domain of the second hybrid polypeptide comprises at least two different retinoid X receptor ligand binding domain fragments selected from the group consisting of a vertebrate species retinoid X receptor ligand binding domain fragment, an invertebrate species retinoid X receptor ligand binding domain fragment, and a non-Dipteran/non-Lepidopteran invertebrate species retinoid X receptor homolog ligand binding domain fragment.

7 - 13 . (canceled)

14 . 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 hybrid 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 chimeric retinoid X receptor ligand binding domain; 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 hybrid polypeptide comprising:

i) a transactivation domain; and

ii) an ecdysone receptor ligand binding domain.

15 . (canceled)

16 . The gene expression modulation system according to claim 14 , wherein the chimeric retinoid X receptor ligand binding domain of the first hybrid polypeptide comprises at least two different retinoid X receptor ligand binding domain fragments selected from the group consisting of a vertebrate species retinoid X receptor ligand binding domain fragment, an invertebrate species retinoid X receptor ligand binding domain fragment, and a non-Dipteran/non-Lepidopteran invertebrate species retinoid X receptor homolog ligand binding domain fragment.

17 - 25 . (canceled)

26 . A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a DNA-binding domain and a chimeric retinoid X receptor ligand binding domain.

27 . The gene expression cassette according to claim 26 , wherein the chimeric retinoid X receptor ligand binding domain comprises at least two different retinoid X receptor ligand binding domain fragments selected from the group consisting of a vertebrate species retinoid X receptor ligand binding domain fragment, an invertebrate species retinoid X receptor ligand binding domain fragment, and a non-Dipteran/non-Lepidopteran invertebrate species retinoid X receptor homolog ligand binding domain fragment.

28 - 31 . (canceled)

32 . A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a transactivation domain and a chimeric retinoid X receptor ligand binding domain.

33 - 35 . (canceled)

36 . An isolated polynucleotide selected from the group consisting of:

(a) a polynucleotide encoding a truncated chimeric retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation reduces ligand binding activity of the truncated chimeric retinoid X receptor ligand binding domain;

(b) a polynucleotide encoding a truncated chimeric retinoid X receptor binding domain comprising a truncation mutation, wherein the truncation mutation reduces steroid binding activity of the truncated chimeric retinoid X receptor ligand binding domain;

(c) a polynucleotide encoding a truncated chimeric retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation reduces non-steroid binding activity of the truncated chimeric retinoid X receptor ligand binding domain;

(d) a polynucleotide encoding a truncated chimeric retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation enhances ligand binding activity of the truncated chimeric retinoid X receptor ligand binding domain;

(e) a polynucleotide encoding a truncated chimeric retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation enhances steroid binding activity of the truncated chimeric retinoid X receptor ligand binding domain;

(f) a poly nucleotide encoding a truncated chimeric retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation enhances non-steroid binding activity of the truncated chimeric retinoid X receptor ligand binding domain;

(g) a polynucleotide encoding a truncated chimeric retinoid X receptor ligand and binding domain comprising a truncation mutation, wherein the truncation mutation increases ligand sensitivity of the truncated chimeric retinoid X receptor ligand binding domain; and

(h) a polynucleotide encoding a truncated chimeric retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation increases ligand sensitivity of a heterodimer, wherein the heterodimer comprises the truncated chimeric retinoid X receptor ligand binding domain and a dimerization partner.

37 - 44 . (canceled)

45 . An isolated polynucleotide encoding a chimeric retinoid X receptor ligand binding domain, wherein the polynucleotide comprises a nucleic acid sequence selected from the group consisting of

a) nucleotides 1-348 of SEQ ID NO: 13 and nucleotides 268-630 of SEQ ID NO: 21,

b) nucleotides 1-408 of SEQ ID NO: 13 and nucleotides 337-630 of SEQ ID NO: 21,

c) nucleotides 1-465 of SEQ ID NO: 13 and nucleotides 403-630 of SEQ ID NO: 21,

d) nucleotides 1-555 of SEQ ID NO: 13 and nucleotides 490-630 of SEQ ID NO: 21,

e) nucleotides 1-624 of SEQ ID NO: 13 and nucleotides 547-630 of SEQ ID NO: 21,

f) nucleotides 1-645 of SEQ ID NO: 13 and nucleotides 601-630 of SEQ ID NO: 21, and

g) nucleotides 1-717 of SEQ ID NO: 13 and nucleotides 613-630 of SEQ ID NO: 21.

46 . (canceled)

47 . An isolated chimeric retinoid X receptor polypeptide comprising an amino acid sequence selected from the group consisting of

a) amino acids 1-116 of SEQ ID NO: 19 and amino acids 90-210 of SEQ ID NO: 27,

b) amino acids 1-136 of SEQ ID NO: 19 and amino acids 113-210 of SEQ ID NO: 27,

c) amino acids 1-155 of SEQ ID NO: 19 and amino acids 135-210 of SEQ ID NO: 27,

d) amino acids 1-185 of SEQ ID NO: 19 and amino acids 164-210 of SEQ ID NO: 27,

e) amino acids 1-208 of SEQ ID NO: 19 and amino acids 183-210 of SEQ ID NO: 27,

f) amino acids 1-215 of SEQ ID NO: 19 and amino acids 201-210 of SEQ ID NO: 27, and

g) amino acids 1-239 of SEQ ID NO: 19 and amino acids 205-210 of SEQ ID NO: 27.

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;

wherein the gene to be modulated is a component of a gene expression cassette comprising:

i) a response element recognized by the DNA binding domain from the first hybrid polypeptide binds;

ii) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and

iii) a gene whose expression is to be modulated;

whereby upon introduction of the ligand into the host cell, expression of the gene of b)iii) is modulated.

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 N, 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 . (canceled)

51 . 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 14 ; and

b) introducing into the host cell a ligand;

wherein the gene to be modulated is a component of a gene expression cassette comprising:

i) a response element recognized by the DNA binding domain from the first hybrid polypeptide;

ii) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and

iii) a gene whose expression is to be modulated;

whereby upon introduction of the ligand into the host cell, expression of the gene of b)iii) is modulated.

52 . The method according to claim 51 , 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 N, 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.

53 . (canceled)

54 . An isolated host cell comprising the gene expression modulation system according to claim 1 .

55 - 56 . (canceled)

57 . An isolated host cell comprising the gene expression modulation system according to claim 14 .

58 - 59 . (canceled)

60 . A non-human organism comprising the host cell of claim 54 .

61 - 62 . (canceled)

63 . A non human organism comprising the host cell of claim 57 .

64 - 65 . (canceled)

66 . The non-human organism of claim 60 , wherein said non-human organism is a non-transgenic organism.

67 . The non-human organism of claim 63 , wherein said non-human organism is a non-transgenic organism.