TRANSGENIC ANIMAL MODELS USING YACS

THE GOAL OF THE PROGRAM IS TO DEVELOP METHODS FOR THE INTRODUCTION OF YEAST ARTIFICIAL CHROMOSOMES (YACS) INTO THE GERMLINE OF MICE, AND TO APPLY THE TECHNOLOGY TOWARD THE GENERATIONOF TRANSGENIC MICE EXPRESSING A BROAD, FULLY HUMAN IMMUNOGLOBULIN REPERTOIRE. THESE MICE WOULDPERMIT THE ISOLATION OF HUMAN MONOCLONAL ANTIBODIES IN AN ANALOGOUS MANNER TO RODENT MONOCLONALANTIBODIES. THE GOAL OF PHASE I IS TO INTRODUCE SMALL YACS CARRYING V,D,J AND CMU REGIONS OF THE HUMAN HEAVY CHAIN IMMUNOGLOBULIN MINIGENE. THE MICE WILL BE TESTED FOR EXPRESSION OF HUMAN IGM PROTEINS IN THE SERUM, AND FOR DNA REARRANGEMENT IN SPLEEN B-CELLS. IN PHASE II, THEANIMALS GENERATED IN PHASE I WILL BE BRED WITH HUMAN KAPPA LIGHT CHAIN EXPRESSING TRANSGENIC MICE, AND THE RESULTANT PILOT MOUSE TESTED FOR ITS CAPACITY TO EXPRESS A PRIMARY REPERTOIRE OF SURFACE AND SECRETED FULLY HUMAN IGMS. ALSO, ADDITIONAL CONSTANT AND VARIABLE REGIONS WILL BE CLONED IN YACS TO ADD TO OR REPLACE THE PHASE I YACS, TO PERMIT CLASS SWITCHING, SOMATIC MUTATION AND IGG PRODUCTION. IF NECESSARY, SEQUENCE MODIFICATIONS SUCH AS THE INTRODUCTION OF ADDITIONAL ENHANCER ELEMENTS WILL ALSO BE ADDED. TRANSGENIC MICE WILL ALSO BE GENERATED CARRYING LARGE FRAGMENTS OF THE UNREARRANGED HUMAN KAPPA GENE AND BRED TO THE PHASE II HEAVY CHAIN MICE. FINALLY, THESE MICE WILL BE BRED WITH MICE LACKING FUNCTIONAL ENDOGENOUS IMMUNOGLOBULIN GENES. HYBRIDOMAS WILL BE GENERATED AT VARIOUS STAGES OF THE PROJECT FROM THEIROFFSPRING TO DETERMINE IF SOMATIC MUTATION OF THE TRANSGENE CAN EFFICIENTLY OCCUR, LEADING TO A BROAD REPERTOIRE OF HIGH AFFINITY, FULLY HUMAN IGG PRODUCING B CELLS.