REPORTER GENE VISUALIZATION IN RHESUS EMBRYOS

University Of Wisconsin Madison, Madison WI

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Abstract

OBJECTIVE To monitor transgene expression in living rhesus embryos. Because of the relative inefficiency of transgene expression in mammalian embryos and the modest numbers (compared to mice) of embryos available on a routine basis, we need to develop approaches to maximize efficiency before transgene delivery to nonhuman primate embryos becomes practical. For example, we will need to insure that embryos transferred to recipient dams have a high likelihood of transgene expression. We have been working with Epstein-Barr Virus-based vectors, which are stably expressed in the nucleus rather than being integrated into the host cell genome. Cycling female rhesus monkeys receive injections of recombinant human FSH and CG to stimulate the growth and maturation of ovarian follicles. Oocytes are fertilized in vitro, and pronuclei are microinjected the day after fertilization. The constructs injected contained either the human CMV intermediate early promoter or the hCG alpha subunit promoter, cloned upstream of an encephalomyelitis virus internal ribosomal entry site (IRES) which is followed by a cDNA for the jellyfish green fluorescent protein (GFP). Using cultured trophoblasts, we have confirmed that GFP expression is driven efficiently off the IRES. Microinjected embryos were subjected to daily illumination to verify transgene expression. We have obtained 25-50% of microinjected embryos expressing the GFP transgene as early as the 4-cell stage, and GFP expression is consistently carried through subsequent in vitro development up to a week following fertilization. Expression is seen in multiple blastomeres throughout development. These results demonstrate that the basic construct approach is sound, and that the EBV-based vectors are effectively replicated during in vitro development. FUTURE DIRECTIONS We will develop highly efficient surgical and nonsurgical embryo transfer methods to rhesus recipients. KEY WORDS placenta, transgene, in vitro fertilization, green fluorescent protein FUNDING NIH HD 26458, RR00167