Chimeric retinoid X receptors and their use in a novel ecdysone receptor-based inducible gene expression system
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 traits in transgenic organisms.
1. A gene expression cassette comprising a polynucleotide encoding a chimeric polypeptide comprising a transactivation domain and a chimeric retinoid X receptor ligand binding domain, wherein at least one alpha helix of said chimeric retinoid X receptor ligand binding domain is replaced with an alpha helix from a ligand binding domain of a retinoid X receptor ligand binding domain of a different species.
2. The gene expression cassette according to claim 1 , wherein the transactivation domain is a VP 16 transactivation domain or a B42 acidic activator transactivation domain.
3. The gene expression cassette according to claim 1 , wherein the gene expression cassette comprises a polynucleotide sequence encoding a chimeric polypeptide, said chimeric polypeptide comprising a transactivation domain encoded by a polynucleotide comprising SEQ ID NO: 51or SEQ ID NO: 53 and wherein said chimeric polypeptide also comprises a chimeric retinoid X receptor ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of
(a) SEQ ID NO: 45,
(b) nucleotides 1-348 of SEQ ID NO: 13 and nucleotides 268-630 of SEQ ID NO: 21,
(c) nucleotides 1-408 of SEQ ID NO: 13 and nucleotides 337-630 of SEQ ID NO: 21,
(d) nucleotides 1-465 of SEQ ID NO: 13 and nucleotides 403-630 of SEQ ID NO: 21,
(e) nucleotides 1-555 of SEQ ID NO: 13 and nucleotides 490-630 of SEQ ID NO: 21,
(f) nucleotides 1-624 of SEQ ID NO: 13 and nucleotides 547-630 of SEQ ID NO: 21,
(g) nucleotides 1-645 of SEQ ID NO: 13 and nucleotides 601-630 of SEQ ID NO: 21, and
(h) nucleotides 1-717 of SEQ ID NO: 13 and nucleotides 613-630 of SEQ ID NO: 21.
4. The gene expression cassette according to claim 1 , wherein the gene expression cassette comprises a polynucleotide sequence encoding a chimeric polypeptide comprising a transactivation domain comprising SEQ ID NO: 52 or SEQ ID NO: 54, and wherein said chimeric polypeptide also comprises a chimeric retinoid X receptor ligand binding domain comprising an amino acid sequence selected from the group consisting of
(a) SEQ ID NO: 46,
(b) amino acids 1-116 of SEQ ID NO: 19 and amino acids 90-210 of SEQ ID NO: 27,
(c) amino acids 1-136 of SEQ ID NO: 19 and amino acids 113-210 of SEQ ID NO: 27,
(d) amino acids 1-155 of SEQ ID NO: 19 and amino acids 135-210 of SEQ ID NO: 27,
(e) amino acids 1-185 of SEQ ID NO: 19 and amino acids 164-210 of SEQ ID NO: 27,
(f) amino acids 1-208 of SEQ ID NO: 19 and amino acids 183-210 of SEQ ID NO: 27,
(g) amino acids 1-215 of SEQ ID NO: 19 and amino acids 201-210 of SEQ ID NO: 27, and
(h) amino acids 1-239 of SEQ ID NO: 19 and amino acids 205-210 of SEQ ID NO: 27.
5. The gene expression cassette according to claim 1 , wherein said alpha helix of said retinoid X receptor ligand binding domain from the different species is a non-Dipteran/non-Lepidopteran invertebrate species retinoid X receptor ligand binding domain alpha helix.
6. The gene expression cassette according to claim 5 , wherein inclusion of said alpha helix of said retinoid X receptor ligand binding domain from a different species results in improved gene expression in mammalian cells.
7. The gene expression cassette according to claim 1 , wherein said chimeric retinoid X receptor ligand binding domain comprises at least one invertebrate species retinoid X receptor alpha helix and at least one vertebrate species retinoid X receptor alpha helix.
8. The gene expression cassette according to claim 7 , wherein said at least one vertebrate species retinoid X receptor alpha helix is selected from the group consisting of EF-domain helices 1-6, EF-domain helices 1-7, EF-domain helices 1-8, EF-domain helices 1-9, EF-domain helices 1-10, and EF-domain helices 1-11, wherein said vertebrate species is mammalian.
9. A vector comprising the gene expression cassette according to claim 1 .
10. The gene expression cassette according to claim 1 , wherein said chimeric retinoid X receptor ligand binding domain comprises at least one vertebrate retinoid X receptor alpha helix.