The nonhuman primate remains the most relevant model for many aspects of human physiology and disease, including reproduction and pregnancy, virology and immunology, higher brain functions including emotion, and the biology of aging. Yet, the primate lags behind rodents in experimental genetic manipulation, e.g., transgenesis. Very recent advances in the manipulation of primate reproduction (ovarian stimulation, embryo development in vitro) have changed the landscape of what is feasible with the nonhuman primate model. In this application, we propose four Specific Aims to develop approaches for the achievement of transgenic primates.
Specific Aim 1 is to deliver transgenes to rhesus monkey preimplantation embryos with Epstein-Barr Virus (EBV)-based and retrovirus-based vectors.
Specific Aim 2 is to establish nonsurgical embryo transfer methods for the rhesus monkey.
Specific Aim 3 is to define beta-galactosidase transgene expression in the rhesus placenta directed by trophoblast-specific promoters, and in the neonate by ubiquitously expressed housekeeping promoters.
Specific Aim 4 is to develop conditional expression of transgenes in vitro and in vivo with a tetracycline-based regulatory system. The proposed studies will employ a mammalian """"""""housekeeping"""""""" or strong viral promoter to direct a model transgene (beta-galactosidase) coupled to a reporter which can be monitored noninvasively (green fluorescent protein, GFP). GFP will allow us to readily visualize successful transduction of embryos, and beta-gal is an easily visualized transgene in the fetus and neonate. We will use the chorionic gonadotropin alpha subunit promoter to direct transgene expression to the placenta as a model system for the tissue-specific expression of transgenes. The development of a general approach to manipulate placental function would not only demonstrate the feasibility of the method, but would have great significance for furthering our understanding of maternal-fetal interactions, including implantation, preeclampsia, intrauterine growth retardation, premature rupture of the membranes and preterm labor. Availability of a transgenic primate resource will promote the creative use of the nonhuman primate model, and will have vast overall significance in the study of human disease and primate experimental embryology, with relevance for other studies with rhesus embryonic stem cells and rhesus embryo clonin for the derivation of MHC-identical monkeys for virology and vaccine development.
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