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.
Claims31
A method of modulating the expression of a gene in a host cell comprising:
- (a) introducing into the host cell a polynucleotide encoding a gene expression modulation system comprising a transactivation domain, a DNA binding domain and a Group H nuclear receptor ligand binding domain that is capable of binding a diacylhydrazine and comprises an amino acid residue substitution mutation at
- (i) at least one of amino residues 54, 119, and 132 of SEQ ID NO:
- 1,
- (ii) both of amino acid residues 96 and 119 of SEQ ID NO:
- 1,
- (iii)
- both of amino acid residues 52 and 110 of SEQ ID NO:
- 1,
- (iv) both of amino acid residues 132 and 125 of SEQ ID NO:
- 1,
- (v) all three of amino acid residues 107, 110 and 127 of SEQ ID NO:
- 1,
- (vi) all three of amino acid residues 52, 107 and 127 of SEQ ID NO:
- 1,
- (vii) both of amino acid residues 107, 127 and an insertion at 259 of SEQ ID NO:
- 1, or
- (viii) an amino acid substitution at least one of the mutations F48Y, F48W, F48L, F48N, F48R, F48K, I51M, I51N, I51L, T52M, T52E, T52P, T52R, T52W, T52G, T52Q, M92L, M92E, R95H, R95M, R95W, V96L, V96W, V96S, V96E, F109W, F109P, F109L, F109M, F109N, Y120W, Y120M, M219K, M219W, M219Y, M219A, L223K, L223R, L223Y, L234M, L234I, L234R, L234W, W238P, W238E, W238Y, W238M and W238L of SEQ ID NO:
- 1;
- said gene expression modulation system further comprising a gene expression cassette comprising:
- (i) a response element recognized by the DNA binding domain;
- (ii) a promoter that is activated by the transactivation domain;
- and
- (iii) a gene whose expression is to be modulated;
- and
- (b) introducing into the host cell a ligand;
- whereby upon introduction of the ligand into the host cell, expression of the gene whose expression is to be modulated is induced, compared to the expression of said gene by a gene expression modulation system in which the same Group H nuclear receptor ligand binding domain does not contain said mutation.
A method of modulating the expression of a gene in a host cell comprising:
- (a) introducing into the host cell a polynucleotide encoding a gene expression modulation system comprising a transactivation domain, a DNA binding domain and a Group H nuclear receptor ligand binding domain that is capable of binding a diacylhydrazine and comprises an amino acid substitution mutation at amino acid residue 132 of SEQ ID NO:
- 1, said gene expression modulation system further comprising a gene expression cassette comprising:
- (i) a response element recognized by the DNA binding domain;
- (ii) a promoter that is activated by the transactivation domain;
- and
- (iii) a gene whose expression is to be modulated;
- and
- (b) introducing into the host cell a ligand;
- whereby upon introduction of the ligand into the host cell, expression of the gene whose expression is to be modulated is induced, compared to the expression of said gene by a gene expression modulation system in which the same Group H nuclear receptor ligand binding domain does not contain said mutation.
Dependencies are inferred from the claim text.