Ligand-regulable transactivation systems, methods of use thereof, methods of detecting estrogen receptor ligands, and methods of differentiating estrogen receptor ligand agonists and antagonists

A method of detecting a ligand in vivo, comprising:

• implanting cells into a nude mouse, wherein the cells comprise exogenous polynucleotides expressing a ligand-regulable transactivation system comprising:
• a reporter polynucleotide that comprises a GAL 4 binding sequence, a promoter sequence, and a reporter sequence encoding a detectable bioluminescent protein, wherein the binding sequence is connected with the promoter sequence and the promoter sequence is connected with the reporter sequence;
• and
• an activator fusion protein that comprises:
• a DNA binding domain capable of interacting with the binding sequence of the reporter polynucleotide,
• an estrogen receptor folding domain (ER folding domain) capable of binding a ligand, wherein the ER folding domain is selected from:
• SEQ ID NO:23 and SEQ ID NO:24, and
• a VP16 transactivation domain capable of interacting with the promoter sequence of the reporter polynucleotide,
• wherein the DNA binding domain is connected to the estrogen receptor folding domain, and the estrogen receptor folding domain is connected with the transactivation domain, and wherein interaction of the DNA binding domain and the transactivation domain of the activator fusion protein with the binding sequence and promoter sequence of the reporter polynucleotide regulates expression of the detectable bioluminescent protein;
• introducing a ligand to the animal, wherein the ligand is not an endogenous ligand in the mouse;
• and
• detecting a bioluminescent signal in the presence of a bioluminescence initiating compound if the ligand causes the ER folding domain to change from a non-interacting conformational position to an interacting conformational position, wherein the interacting conformational position positions the DNA binding domain and the transactivation domain of the activator fusion protein so that both interact with the binding sequence and the promoter sequence of the reporter polynucleotide, which causes expression of the detectable bioluminescent protein encoded by the reporter sequence.

A method of detecting a ligand in vitro, comprising:

• providing a ligand-regulable transactivation system comprising:
• a reporter polynucleotide that comprises a GAL4 binding sequence of SEQ ID NO:17, a promoter sequence, and a reporter sequence encoding a detectable bioluminescent protein, wherein the binding sequence is connected with the promoter sequence and the promoter sequence is connected with the reporter sequence;
• and
• an activator fusion protein that comprises:
• a DNA binding domain capable of interacting with the GAL4 binding sequence of the reporter polynucleotide,
• an estrogen receptor folding domain (ER folding domain) capable of binding a ligand, and
• a transactivation domain capable of interacting with the promoter sequence of the reporter polynucleotide,
• wherein the DNA binding domain is connected to the estrogen receptor folding domain, and the estrogen receptor folding domain is connected with the transactivation domain, and wherein interaction of the DNA binding domain and the transactivation domain of the activator fusion protein with the binding sequence and promoter sequence of the reporter polynucleotide regulates expression of the detectable bioluminescent protein;
• introducing a ligand to the system;
• and
• detecting a bioluminescent signal in the presence of a bioluminescence initiating compound if the ligand causes the ER folding domain to change from a non-interacting conformational position to an interacting conformational position, wherein the interacting conformational position positions the DNA binding domain and the transactivation domain of the activator fusion protein so that both interact with the binding sequence and the promoter sequence of the reporter polynucleotide, which causes expression of the detectable bioluminescent protein encoded by the reporter sequence.