PREIMPLANTATION IDENTIFICATION OF TRANSGENIC EMBRYOS

THE OVERALL GOAL OF THIS STUDY IS TO EVALUATE THE USE OF EMBRYONIC BIOPSY AND POLYMERASE CHAIN REACTION (PCR) AMPLIFICATION TO IDENTIFY MICROINJECTED EMBRYOS IN WHICH THE FOREIGN DNA HAS BEEN INTEGRATED INTO THE GENOME. MICROINJECTED PRONUCLEAR EMBRYOS WILL BE CULTURED TO THE EIGHT-CELL STAGE, AND A SMALL SAMPLE OF EMBRYONIC CELLS WILLBE REMOVED WITH A BIOPSY PIPET AND LYSED TO PROVIDE A SMALL SAMPLE OF DNA FOR ANALYSIS. THIS METHOD OF GENOMIC ANALYSIS WILL MAINTAIN EMBRYO VIABILITY AND IS INTENDED TO RESTRICT EMBRYO TRANSFER TO THESMALL SUBSET OF MICROINJECTED EMBRYOS BEARING INTEGRATED TRANSGENES. PCR RESULTS OBTAINED FROM LYSATES OF THE BIOPSYSAMPLES WILL BE VALIDATED BY SPECIFIC RESTRICTION ENZYME DIGESTS DESIGNED TO DISCRIMINATE BETWEEN INTEGRATED AND NONINTEGRATED TRANSGENES AND BY ANALYSIS OF OFFSPRING. THESE TECHNIQUES WILL BE DEVELOPED AND EVALUATED IN PHASE I USING A MOUSE MODEL SYSTEM. THE METHODS RESULTING FROM THE PHASE I STUDY WILL BE UTILIZED IN PHASE II OF THE PROJECT, IN WHICH A HERD OF TRANSGENIC DAIRY GOATS, EXPRESSING MAMMARY-SPECIFIC TRANSGENES, WILL BE DEVELOPED FOR USE AS PHARMACEUTICAL BIOREACTORS.