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.
Claims32
A recombinant gene expression modulation system comprising a recombinant gene expression cassette that is capable of being expressed in a host cell, the gene expression cassette comprising a polynucleotide that encodes a first polypeptide comprising:
- (a) a transactivation domain;
- (b) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated;
- and
- (c) a nuclear receptor ligand binding domain that is capable of binding a diacylhydrazine and comprises either an amino acid residue substitution mutation of SEQ ID NO:
- 1 at
- (i) at least one of amino residues 54, 119, 127, 128 and 132,
- (ii) amino acid residues 96 and 119,
- (iii) amino acid residues 110 and 128,
- (iv) amino acid residues 52 and 110,
- (v) amino acid residues 132 and 125
- (vi) amino acid residues 107, 110 and 127,
- (vii) amino acid residues 52, 107 and 127, or
- (viii) amino acid residues 107, 127 and an insertion at 259, or
- at least one of the mutations F48Y, F48W, F48L, F48N, F48R, F48K, I51M, I51N, 151L, T52M, T52E, T52P, T52R, T52W, T52G, T52Q, M92L, M92E, R95H, R95M, R95W, V96L, V96W, V96S, V96E, V96T, V1071, F109W, F109P, F109L, F109M, F109N, A110E, A110N, A110P, A110W, Y120W, Y120M, M125P, M125R, M125E, M125L, M125C, M125W, M125G, M125I, M125N, M125S, M125V, Y127E, M219K, M219W, M219Y, M219A, L223K, L223R, L223Y, L234M, L2341, L234R, L234W, W238P, W238E, W238Y, W238M and W238L.
A recombinant gene expression modulation system comprising:
- (a) a first recombinant 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 first nuclear receptor ligand binding domain;
- and
- (b) a second recombinant 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 second nuclear receptor ligand binding domain,
- wherein one of said first and said second nuclear receptor ligand binding domains is capable of binding a diacylhydrazine, and comprises either an amino acid residue substitution mutation of SEQ ID NO:
- 1 at
- (1) at least one of amino residues 54, 119, 127, 128 and 132,
- (2) amino acid residues 96 and 119,
- (3) amino acid residues 110 and 128,
- (4) amino acid residues 52 and 110,
- (5) amino acid residues 132 and 125
- (6) amino acid residues 107, 110 and 127,
- (7) amino acid residues 52, 107 and 127, or
- (8) amino acid residues 107, 127 and an insertion at 259, or
- at least one of the mutations F48Y, F48W, F48L, F48N, F48R, F48K, I51M, I51N, 151L, T52M, T52E, T52P, T52R, T52W, T52G, T52Q, M92L, M92E, R95H, R95M, R95W, V96L, V96W, V96S, V96E, V96T, V107I, F109W, F109P, F109L, F109M, F109N, A110L, A110N, A110P, A110W, Y120W, Y120M, M125P, M125R, M125E, M125L, M125C, M12SW, M125G, M125I, M125N, M125S, M125V, Y127E, M219K, M219W, M219Y, M219A, L223K, L223R, L223Y, L234M, L234I, L234R, L234W, W238P, W238E, W238Y, W238M and W238L.
An isolated polynucleotide, wherein said isolated polynucleotide encodes a nuclear receptor ligand binding domain that is capable of binding a diacylhydrazine, and comprises either an amino acid residue substitution mutation of SEQ ID NO:
- 1 at
- (a) at least one of amino residues 54, 119, 127, 128 and 132,
- (b) amino acid residues 96 and 119,
- (c) amino acid residues 110 and 128,
- (d) amino acid residues 52 and 110,
- (e) amino acid residues 132 and 125
- (f) amino acid residues 107, 110 and 127,
- (g) amino acid residues 52, 107 and 127, or
- (h) amino acid residues 107, 127 and an insertion at 259, or
- at least one of the mutations F48Y, F48W, F48L, F48N, F48R, F48K, 151M, 151N, I51L, T52M, T52E, T52P, T52R, T52W, T52G, T52G, M92L, M92E, R95H, R95M, R95W, V96L, V96W, V96S, V96E, V96T, V107I, F109W, F109P, F109L, F109M, F109N, A110E, A110N, A110P, A110W, Y120W, Y120M, M125P, M125R, M125E, M125L, M125C, M125W, M125G, M125I, M125N, M125S, M125V, Y127E, V128F, M219K, M219W, M219Y, M219A, L223K, L223R, L223Y, L234M, L2341, L234R, L234W, W238P, W238E, W238Y, W238M and W238L.
A recombinant gene expression cassette comprising an isolated polynucleotide that encodes
- (a) a polypeptide comprising a transactivation domain, a DNA-binding domain, and a nuclear receptor ligand binding domain,
- (b) a polypeptide comprising a DNA-binding domain and a nuclear receptor ligand binding domain, or
- (c) a polypeptide comprising a transactivation domain and a nuclear receptor ligand binding domain,
- wherein said nuclear receptor ligand binding domain is capable of binding a diacylhydrazine, and comprises either an amino acid residue substitution mutation of SEQ ID NO:
- 1 at
- (a) at least one of amino residues 54, 119, 127, 128 and 132,
- (b) amino acid residues 96 and 119,
- (c) amino acid residues 110 and 128,
- (d) amino acid residues 52 and 110,
- (e) amino acid residues 132 and 125
- (f) amino acid residues 107, 110 and 127,
- (g) amino acid residues 52, 107 and 127, or
- (h) amino acid residues 107, 127 and an insertion at 259, or
- at least one of the mutations F48Y, F48W, F48L, F48N, F48R, F48K, 151M, 151N, I51L, T52M, T52E, T52P, T52R, T52W, T52G, T520, M92L, M92E, R95H, R95M, R95W, V96L, V96W, V96S, V96E, V96T, V1071, F109W, F109P, F109L, F109M, F109N, A110E, A110N, A110P, A110W, Y120W, Y120M, M125P, M125R, M125E, M125L, M125C, M125W, M125G, M125I, M125N, M125S, M125V, Y127E, V128F, M219K, M219W, M219Y, M219A, L223K, L223R, L223Y, L234M, L234I, L234R, L234W, W238P, W238E, W238Y, W238M and W238L.
Dependencies are inferred from the claim text.