Mutant Receptors and Their Use in a Nuclear Receptor-Based Inducible Gene Expression System
AssigneeIntrexon Corporation
InventorsSubba Reddy PALLI, Mohan Basavaraju KUMAR
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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.
Claims21
A gene expression modulation system comprising a gene expression cassette that is capable of being expressed in a host cell, the gene expression cassette comprising a polynucleotide that encodes a polypeptide comprising:
- i) a transactivation domain;
- ii) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated;
- and
- iii) a Group H nuclear receptor ligand binding domain comprising at least one mutation, wherein the mutation is at a position equivalent to or analogous to amino acid residues selected from the group consisting of:
- a) at least one of 48, 51, 52, 54, 92, 95, 96, 109, 110, 119, 120, 125, 128, 132, 219, 223, 234, and 238 of SEQ ID NO:
- 1, b) both 96 and 119 of SEQ ID NO:
- 1, c) both 110 and 128 of SEQ ID NO:
- 1, d) both 52 and 110 of SEQ ID NO:
- 1, e) both 107, 110, and 127 of SEQ ID NO:
- 1, f) both 52, 107 and 127 of SEQ ID NO:
- 1 and g) both 107, 127 and 259 of SEQ ID NO:
- 1, or any combination thereof.
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 that encodes a first 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 nuclear receptor ligand binding domain;
- and
- b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide that encodes a second polypeptide comprising:
- i) a transactivation domain;
- and
- ii) a nuclear receptor ligand, binding domain
- wherein one of the nuclear receptor ligand binding domains is a Group H nuclear receptor ligand binding domain comprising at least one mutation, wherein the mutation is at a position equivalent to or analogous to amino acid residues selected from the group consisting of:
- a) at least one of 48, 51, 52, 54, 92, 95, 96, 109, 110, 119, 120, 125, 128, 132, 219, 223, 234 and 238 of SEQ ID NO:
- 1, b) both 96 and 119 of SEQ ID NO:
- 1, c) both 110 and 128 of SEQ ID NO:
- 1, d) both 52 and 110 of SEQ ID NO:
- 1, e) all three of 107, 110, and 127 of SEQ ID NO:
- 1, f) all three of 52, 107 and 127 of SEQ ID NO:
- 1 and g) all three of 107, 127 and 259 of SEQ ID NO:
- 1, or any combination thereof.
An isolated polynucleotide encoding a Group H nuclear receptor ligand binding domain comprising at least one mutation, wherein the isolated polynucleotide comprises at least one mutation that results in at least one mutation of an amino acid residue at a position equivalent to or analogous to a) at least one of amino acid residue 48, 51, 52, 54, 92, 95, 96, 109, 110, 119, 120, 125, 128, 132, 219, 223, 234, and 238 of SEQ ID NO:
- 1, b) both amino acid residues 96 and 119 of SEQ ID NO:
- 1, c) both amino acid residues 110 and 128 of SEQ ID NO:
- 1, d) both amino acid residues 52 and 110 of SEQ ID NO:
- 1, e) all three amino acid residues 107, 110, and 127 of SEQ ID NO:
- 1, f) all three amino acid residues 52, 107 and 127 of SEQ ID NO:
- 1 or g) all three amino acid residues 107, 127 and 259 of SEQ ID NO:
- 1 or any combination thereof.
An isolated polypeptide comprising a Group H nuclear receptor ligand binding domain comprising at least one mutation, wherein the mutation is at a position equivalent to or analogous to a) at least one of amino acid residue 48, 51, 52, 54, 92, 95, 96, 109, 110, 119, 120, 125, 128, 132, 219, 223, 234, and 238 of SEQ ID NO:
- 1, b) both amino acid residues 96 and 119 of SEQ ID NO:
- 1, c) both amino acid residues 110 and 128 of SEQ ID NO:
- 1, d) both amino acid residues 52 and 110 of SEQ ID NO:
- 1, e) all three amino acid residues 107, 110, and 127 of SEQ ID NO:
- 1, f) all three amino acid residues 52, 107 and 127 of SEQ ID NO:
- 1 or g) all three amino acid residues 107, 127 and 259 of SEQ ID NO:
- 1, or any combination thereof.
Dependencies are inferred from the claim text.