PROTEIN PAINTING OF PREIMPLANTATION EMBRYOS

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DESCRIPTION: (Scanned from the applicant's abstract) The rate of growth of preimplantation embryos is regulated by both environmental and genetic factors and can have a profound effect on the later health of the offspring from birth into adulthood. Using the mouse as a model system, one gene that influences growth rate of preimplantation embryos and pregnancy outcome has been identified, the Ped gene. The product of the Ped gene is Qa-2 protein. This protein is attached to the plasma membrane of embryonic cells by a glycosylphosphatidylinositol (GPI) linkage. Embryos with a fast embryonic growth rate (Pedfast) express GPI-linked Qa-2 protein and have a greater chance of survival to term, as well as higher birth and weaning weights compared to embryos with the absence of GPI-linked Qa-2 protein (Ped slow). Proteins with a GPI linkage can spontaneously incorporate into the plasma membrane of somatic cells (e.g., T cells) in a procedure called "protein painting." In this proposal GPI-linked Qa-2 protein will be used to evaluate whether protein painting can be utilized to add proteins to the cell surface of preimplantation embryos. First, GPI-linked Qa-2 protein will be purified by detergent solubilization followed by affinity chromatography with anti-Qa-2 monoclonal antibody. Next, the purified GPI-linked Qa-2 protein will be used to optimize conditions for painting of preimplantation embryos. Then, the function of the painted Qa-2 protein during the preimplantation period will be assessed by measuring rate of growth and response to crosslinking with anti-Qa-2 antibody. Finally, the effect of the modification of the embryos during the preimplantation period on pregnancy outcome will be determined by evaluating survival to term, birth weight, and weaning weight. Protein painting is a potentially powerful technology because cell surface protein expression can be manipulated without gene transfer. Moreover, any protein could be used for protein painting by splicing the signal to add a GPI anchor to the gene encoding the protein of interest. Protein painting may ultimately be applied to create better conditions for modulating preimplantation embryonic growth or death, leading to enhanced pregnancy outcome or a new contraceptive technology.