GENERATION OF A NOVEL VERSATILE REPORTER FOR THE IN VIVO DETECTION OF GENE EXPRESSION VARIATIONS BY POSITRON EMISSION TOMOGRAPHY (PET)

THIS IS A PROOF-OF-CONCEPT PROPOSAL: TO ESTABLISH WHETHER THE EXPRESSION OF A BACTERIAL B-LACTAMASE CAN BE USED AS A TOOL FOR MONITORING CHANGES IN THE LEVELS OF GENE EXPRESSION IN VIVO. THE SUCCESS OF THIS PROJECT WILL LEAD TO THE GENERATION OF TRANSGENIC ANIMALS OF GREAT VALUE IN PRECLINICAL RESEARCH. IN VIVO NONINVASIVE IMAGING IS THE TOOL OF CHOICE IN PRECLINICAL RESEARCH TO EVALUATE THE EVOLUTION OF DISEASES AND THEIR RESPONSE TO THERAPY. THE OBTAINING OF IN VIVO IMAGES OF GENES BY POSITRON EMISSION TOMOGRAPHY (PET) IS THE TECHNIQUE CHOSEN FOR THIS PURPOSE. THE CURRENT PET IRGS HAVE SEVERAL DRAWBACKS, BEING DIFFICULT TO DETERMINE TO WHAT EXTENT THE SIGNAL DETECTED IS DUE TO A TRANSCRIPTIONAL OR PHOSPHORYLATION CHANGE, INDEPENDENTLY OF THE PROMOTER ACTIVITY AND THE NON-SPECIFICITY DUE TO THE INTERACTION OF THE LABELED LIGAND WITH THE ENDOGENOUS PROTEINS. IN ADDITION, SOME OF THESE CONTROLS ARE HIGHLY IMMUNOGENIC, WHICH LIMITS THEIR USE TO IMMUNO-RESISTANT ANIMALS. TO OVERCOME THESE LIMITATIONS, WE PLAN TO DEVELOP AN IRG SYSTEM BASED ON THE EXPRESSION OF A MODIFIED FORM OF A BACTERIAL B-LACTAMASE. IT IS A MUTANT VARIANT IN THE CATALYTIC CENTER THAT BINDS IRREVERSIBLY TO B-LACTAM ANTIBIOTICS. THE AMOUNTS OF THIS B-LACTAMASE EXPRESSED FROM THE PROMOTER WILL BE QUANTIFIED BY PET, AFTER THE ADMINISTRATION OF AN AMPICILLIN DERIVATIVE (DOTA-AMPICILLIN), WITH HIGH AFFINITY FOR B-LACTAMASE, RADIOACTIVELY LABELED WITH GALLIUM (68GA). ACTION PLAN: 1) GENERATE A STABLE CELL LINE EXPRESSING THE B-LACTAMASE GENE UNDER THE CONTROL OF THE CYTOMEGALOVIRUS (CMV) PROMOTER; 2) INJECTION OF THE POSITIVE B-LACTAMASE CELLS IN MICE TO GENERATE A XENOGRAFT; 3) SYSTEMIC ADMINISTRATION OF 68GALLIUM-AMPICILLIN; AND 4) IMAGE OF THE IN VIVO DISTRIBUTION BY PET.