Novel Substitution Mutant Receptors and Their Use in a Nuclear Receptor-Based Inducible Gene Expression System

A method of modulating the expression of a gene in a host cell comprising the steps of:

• a) introducing into the host cell a gene expression modulation system comprising
• i) a first polynucleotide that encodes a first polypeptide comprising:
• (1) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated;
• and 2) a first nuclear receptor ligand binding domain;
• and
• ii) a second polynucleotide that encodes a second polypeptide comprising:
• 1) a transactivation domain;
• and 2) a second nuclear receptor ligand binding domain;
• wherein one of the first or second nuclear receptor ligand binding domains comprises the amino acid sequence of SEQ ID NO:
• 1 wherein the amino acid sequence of SEQ ID NO:
• 1 has
• i) a single amino acid substitution mutation at amino acid residue 20, 21, 48, 51, 52, 55, 58, 59, 61, 62, 92, 93, 95, 96, 107, 109, 120, 123, 125, 175, 223, 230, 234, or 238 of SEQ ID NO:
• 1;
• or
• ii) a double amino acid substitution at amino acid residues 107 and 175 of SEQ ID NO:
• 1, at amino acid residues 127 and 175 of SEQ ID NO:
• 1, or at amino acid residues 107 and 127 of SEQ ID NO:
• 1;
• or
• iii) a triple amino acid substitution mutation at amino acid residues 107, 127 and 175 of SEQ ID NO:
• 1, at amino acid residues 52, 107 and 175 of SEQ ID NO:
• 1, or at amino acid residues 96, 107 and 175 of SEQ ID NO:
• 1;
• and
• b) introducing into the host cell a ligand;
• wherein the gene to be modulated is a component of 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;
• whereby upon introduction of the ligand into the host cell, expression of the gene of b) iii) is modulated.

A method of modulating the expression of a gene in a host cell comprising the steps of:

• a) introducing into the host cell a gene expression modulation system comprising a gene expression cassette that is capable of being expressed in a host cell 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 nuclear receptor ligand binding domain comprising the amino acid sequence of SEQ ID NO:
• 1 wherein the amino acid sequence of SEQ ID NO:
• 1 has
• 1) a single amino acid substitution mutation at amino acid residue 20, 21, 48, 51, 52, 55, 58, 59, 61, 62, 92, 93, 95, 96, 107, 109, 120, 123, 125, 175, 223, 230, 234, or 238 of SEQ ID NO:
• 1;
• or
• 2) a double amino acid substitution mutation, at amino acid residues 107 and 175 of SEQ ID NO:
• 1, at amino acid residues 127 and 175 of SEQ ID NO:
• 1, or at amino acid residues 107 and 127 of SEQ ID NO:
• 1;
• or
• 3) a triple amino acid substitution mutation at amino acid residues 107, 127 and 175 of SEQ ID NO:
• 1, at amino acid residues 52, 107 and 175 of SEQ ID NO:
• 1, or at amino acid residues 96, 107 and 175 of SEQ ID NO:
• 1,
• b) introducing into the host cell a ligand;
• wherein the gene to be modulated is a component of 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;
• whereby upon introduction of the ligand into the host cell, expression of the gene of b) iii) is modulated.