Non-human organism comprising a gene expression modulation system encoding a chimeric retinoid X receptor
View Patent ↗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 ecdysone receptor/chimeric retinoid X receptor-based 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 organisms.
1. A transgenic, non-human organism comprising a gene expression modulation system comprising:
a) a first gene expression cassette that is capable of being expressed in said organism 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 said organism 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 comprising a vertebrate retinoid X receptor amino acid sequence and an invertebrate retinoid X receptor amino acid sequence,
wherein at least one alpha helix of said vertebrate retinoid X receptor ligand binding domain is replaced with the same number of alpha helices from an invertebrate retinoid X receptor ligand binding domain or the same number of alpha helices from an ultraspiracle (USP) receptor ligand binding domain,
wherein the genome of said non-human organism comprises said gene expression modulation system, and
wherein said system modulates the expression of said gene.
2. The transgenic, non-human organism according to claim 1 , wherein the non-human organism is selected from the group consisting of a bacterium, a fungus, and a yeast.
3. The transgenic, non-human organism according to claim 2 , wherein said organism is a yeast selected from the group consisting of an Aspergillus , a Trichoderma , a Saccharomyces , a Pichia , a Candida , and a Hansenula.
4. The transgenic, non-human organism according to claim 2 , wherein said organism is selected from the group consisting of a Synechocystis , a Synechococcus , a Salmonella , a Bacillus , an Acinetobacter , a Rhodococcus , a Streptomyces , an Escherichia , a Pseudomonas , a Methylomonas , a Methylobacter , an Alcaligenes , an Anabaena , a Thiobacillus , a Methanobacterium , and a Klebsiella.
5. The transgenic, non-human organism according to claim 1 , wherein the organism is a mouse.
6. The transgenic, non-human organism according to claim 1 , wherein the ecdysone receptor ligand binding domain is a Choristoneura fumiferana ecdysone receptor ligand binding domain or a Drosophila melanogaster ecdysone receptor ligand binding domain.
7. The transgenic, non-human organism according to claim 1 , further comprising a third gene expression cassette comprising:
(a) a response element recognized by the DNA-binding domain of the first hybrid polypeptide;
(b) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and
(c) a gene whose expression is to be modulated.
8. The transgenic, non-human organism according to claim 1 , wherein the invertebrate retinoid X receptor amino acid sequence is non-Dipteran/non-Lepidopteran invertebrate retinoid X receptor amino acid sequence.
9. The transgenic, non-human organism according to claim 1 , wherein the chimeric retinoid X receptor ligand binding domain of the second hybrid polypeptide is encoded by a polynucleotide comprising 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.
10. The transgenic, non-human organism according to claim 1 , wherein the chimeric retinoid X receptor ligand binding domain of the second hybrid polypeptide comprises 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.
11. The transgenic, non-human organism according to claim 1 , wherein the DNA-binding domain is selected from a GAL4 DNA-binding domain and a LexA DNA-binding domain.
12. The method transgenic, non-human organism of claim 1 , wherein the transactivation domain is selected from the group consisting of an a VP16 transactivation domain, a B42 acidic activator transactivation domain, a steroid/thyroid hormone nuclear receptor transactivation domain, a polyglutamine transactivation domain, a basic or acidic amino acid transactivation domain, a GAL4 transactivation domain, an NF-κB transactivation domain, and a BP64 transactivation domain.
13. The transgenic, non-human organism according to claim 1 , wherein said ecdysone receptor ligand binding domain is selected from the group consisting of an invertebrate ecdysone receptor ligand binding domain, an Arthropod ecdysone receptor ligand binding domain, a Lepidopteran ecdysone receptor ligand binding domain, a Dipteran 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 beetle Tenebrio molitor ecdysone receptor ligand binding domain, a Manduca sexta ecdysone receptor ligand binding domain, a Heliothies virescens ecdysone receptor ligand binding domain, a midge Chironomus tentans ecdysone receptor ligand binding domain, a silk moth Bombyx mori ecdysone receptor ligand binding domain, a squinting bush brown Bicyclus anynana 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, an ixodid tick Amblyomma americanum ecdysone receptor ligand binding domain, a whitefly Bamecia argentifoli ecdysone receptor ligand binding domain and a leafhopper Nephotetix cincticeps ecdysone receptor ligand binding domain.
14. The transgenic, non-human organism according to claim 1 , wherein said gene expression modulation system is contained in a vector.
15. The transgenic, non-human organism according to claim 14 , wherein said vector is selected from the group consisting of a retrovirus vector, an adeno-associated virus vector, a pox virus vector, a baculovirus vector, a vaccinia virus vector, a herpes simplex virus vector, an Epstein-Barr virus vector, a geminivirus vector, a caulimovirus vector, and an adenovirus vector.
16. The transgenic, non-human organism according to claim 1 , wherein said organism is an animal.
17. The transgenic, non-human organism according to claim 16 , wherein said animal is a mammal.
18. The transgenic, non-human organism according to claim 1 , wherein said mammal is selected from the group consisting of:
a) a rat;
b) a rabbit;
c) a cat;
d) a dog;
e) a bovine;
f) a goat;
g) a pig;
h) a horse;
i) a sheep;
j) a monkey; and,
k) a chimpanzee.
19. A transgenic, non-human organism comprising a gene expression modulation system comprising:
a) a first gene expression cassette that is capable of being expressed in said organism 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 comprising a vertebrate retinoid X receptor amino acid sequence and an invertebrate retinoid X receptor amino acid sequence, wherein at least one alpha helix of said vertebrate retinoid X receptor ligand binding domain is replaced with the same number of alpha helices from an invertebrate retinoid X receptor ligand binding domain or the same number of alpha helices from an ultraspiracle USP receptor ligand binding domain; and
b) a second gene expression cassette that is capable of being expressed in said organism comprising a polynucleotide sequence that encodes a second hybrid polypeptide comprising:
i) a transactivation domain; and
ii) an ecdysone receptor ligand binding domain,
wherein the genome of said non-human organism comprises said gene expression modulation system, and
wherein said system modulates the expression of said gene.
20. The transgenic, non-human organism according to claim 19 , wherein the non-human organism is selected from the group consisting of a bacterium, a fungus, and a yeast.
21. The transgenic, non-human organism according to claim 20 , wherein said organism is a yeast selected from the group consisting of an Aspergillus , a Trichoderma , a Saccharomyces , a Pichia , a Candida , and a Hansenula.
22. The transgenic, non-human organism according to claim 20 , wherein said organism is selected from the group consisting of a Synechocystis , a Synechococcus , a Salmonella , a Bacillus , an Acinetobacter , a Rhodococcus , a Streptomyces , an Escherichia , a Pseudomonas , a Methylomonas , a Methylobacter , an Alcaligenes , an Anabaena , a Thiobacillus , a Methanobacterium , and a Klebsiella.
23. The transgenic, non-human organism according to claim 19 , wherein the organism is a mouse.
24. The transgenic, non-human organism according to claim 19 , wherein the ecdysone receptor ligand binding domain is a Choristoneura fumiferana ecdysone receptor ligand binding domain or a Drosophila melanogaster ecdysone receptor ligand binding domain.
25. The transgenic, non-human organism according to claim 19 , further comprising a third gene expression cassette comprising:
(a) a response element recognized by the DNA-binding domain of the first hybrid polypeptide;
(b) a promoter that is activated by the transactivation domain of the second hybrid polypeptide; and
(c) a gene whose expression is to be modulated.
26. The transgenic, non-human organism according to claim 19 , wherein the invertebrate retinoid X receptor amino acid sequence is non-Dipteran/non-Lepidopteran invertebrate retinoid X receptor amino acid sequence.
27. The transgenic, non-human organism according to claim 19 , wherein the chimeric retinoid X receptor ligand binding domain of the first hybrid polypeptide is encoded by a polynucleotide comprising 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.
28. The transgenic, non-human organism according to claim 19 , wherein the chimeric retinoid X receptor ligand binding domain of the first hybrid polypeptide comprises 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.
29. The transgenic, non-human organism according to claim 19 , wherein the DNA-binding domain is selected from a GAL4 DNA-binding domain and a LexA DNA-binding domain.
30. The transgenic, non-human organism of claim 19 , wherein the transactivation domain is selected from the group consisting of an a VP16 transactivation domain, a B42 acidic activator transactivation domain, a steroid/thyroid hormone nuclear receptor transactivation domain, a polyglutamine transactivation domain, a basic or acidic amino acid transactivation domain, a GAL4 transactivation domain, an NF-κB transactivation domain, and a BP64 transactivation domain.
31. The transgenic, non-human organism according to claim 19 , wherein said ecdysone receptor ligand binding domain is selected from the group consisting of an invertebrate ecdysone receptor ligand binding domain, an Arthropod ecdysone receptor ligand binding domain, a Lepidopteran ecdysone receptor ligand binding domain, a Dipteran 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 beetle Tenebrio molitor ecdysone receptor ligand binding domain, a Manduca sexta ecdysone receptor ligand binding domain, a Heliothies virescens ecdysone receptor ligand binding domain, a midge Chironomus tentans ecdysone receptor ligand binding domain, a silk moth Bombyx mori ecdysone receptor ligand binding domain, a squinting bush brown Bicyclus anynana 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, an ixodid tick Amblyomma americanum ecdysone receptor ligand binding domain, a whitefly Bamecia argentifoli ecdysone receptor ligand binding domain and a leafhopper Nephotetix cincticeps ecdysone receptor ligand binding domain.
32. The transgenic, non-human organism according to claim 19 , wherein said gene expression modulation system is contained in a vector.
33. The transgenic, non-human organism according to claim 32 , wherein said vector is selected from the group consisting of a retrovirus vector, an adeno-associated virus vector, a pox virus vector, a baculovirus vector, a vaccinia virus vector, a herpes simplex virus vector, an Epstein-Barr virus vector, a geminivirus vector, a caulimovirus vector, and an adenovirus vector.
34. The transgenic, non-human organism according to claim 19 , wherein said organism is an animal.
35. The transgenic, non-human organism according to claim 19 , wherein said animal is a mammal.
36. The transgenic, non-human organism according to claim 19 , wherein said mammal is selected from the group consisting of:
a) a rat;
b) a rabbit;
c) a cat;
d) a dog;
e) a bovine;
f) a goat;
g) a pig;
h) a horse;
i) a sheep;
j) a monkey; and,
k) a chimpanzee.