DEVELOPMENT OF SYSTEM TO DETECT PEROXISOME PROLIFERATORS

WE WILL DEVELOP A NOVEL SYSTEM FOR DETECTING CHEMICALS WHICH INDUCE PEROXISOME PROLIFERATION. THIS SYSTEM WILL CONSIST OF A HUMAN CELL LINE WHICH HAS BEEN TRANSFECTED WITH SPECIFIC RECEPTOR AND REPORTER GENE CONSTRUCTS. CELLS WILL BE TRANSFECTED WITH CHIMERIC RECEPTORS CONTAINING THE LIGAND BINDING REGION OF THE PEROXISOME PROLIFERATOR RECEPTOR AND THE DNA BINDING REGION OF THE HUMAN GLUCOCORTICOID RECEPTOR. THE CNDA FOR THE REPORTER IS COUPLED TO A GLUCOCORTICOID RESPONSIVE PROMOTER. THE REPORTER GENE (CDNA) ENCODES AN ENZYME FOR WHICH THERE IS A DIRECT FLUOROMETRIC METHOD FOR DETERMINING CATALYTIC ACTIVITY. THE FLUOROMETRIC METHOD FOR DETERMINING CATALYTIC ACTIVITY. THE FLUOROMETRIC METHOD FOR CATALYTIC ACTIVITY IS SENSITIVE AND CAN BE PERFORMED IN WHOLE CELLS. THE RESULTING ASSAY SYSTEM SHOULD BE EASILY AMMENDABLE TO AUTOMATION FOR SCREENING LARGE NUMBERS OF CHEMICALS.