Transgenic mice have provided invaluable information about gene function and regulation. However, because of marked differences between rodents and primates, some areas of human biology such as early embryonic development, aging, and maternal-fetal interactions would be best studied in a nonhuman primate model. Here, we report that gene transfer into rhesus monkey (Macaca mulatta) preimplantation embryos gives rise to transgenic placentas that express a reporter transgene (eGFP). Blastocysts resulting from culture of in vitro fertilized ova were transduced with a selfinactivating lentiviral vector and transferred into recipient females. One twin and one singleton pregnancy were produced from a single stimulation cycle, and one live rhesus monkey was born from each pregnancy. Placentas from all conceptuses showed expression of the transgene as detected by reverse transcription-PCR, ribonuclease protection assay, direct epifluorescence, immunohistochemistry, and Western blot analysis. Integration in somatic tissues of the offspring was not detected. A maternal immune response to the xenogeneic placental antigen was shown by the presence of anti-GFP antibodies in peripheral blood of the recipient females by day 99 of gestation (term ‫؍‬ 165 days). These results demonstrate that transgene expression during gestation is compatible with successful pregnancy in nonhuman primates and provides an approach that could be broadly applicable to the development of novel models for primate biomedical research.

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The birth of a rhesus monkey resulting from in vitro fertilization is reported. Oocytes recovered at laparoscopy from five gonadotropin-stimulated donors were inseminated in vitro with sperm preincubated with caffeine and dibutyryl cyclic AMP. After insemination, oocytes were cultured for 33-46 hr. Twenty-two embryos were transferred nonsurgically into 11 recipient females. One recipient showed signs of implantation but did not carry to term. A second female became pregnant after receiving one 4-cell and one 6-cell embryo fertilized in vitro. The subsequent course of early pregnancy and embryonic and fetal development were characteristic of normal singleton pregnancies. A healthy term male infant was delivered by Cesarean section 176 days after fertilization. This birth has validated our procedures for in vitro fertilization of rhesus monkey gametes and provides an experimental model for studies of early embryonic development in primates.There is a long-standing need for models using nonhuman primates to supply basic information on fertilization and early embryogenesis. This need has been increased by the clinical applications of in vitro fertilization (IVF) We have devised procedures for IVF and embryo culture using the rhesus monkey (6). In this species, the average duration of the menstrual cycle (28 days), the regulation of folliculogenesis, and the control of ovulation are all very similar to these processes in women (7). Because the rhesus has served as a model for early human development, extensive data on postnatal behavior and development are available (8). Thus, even subtle developmental anomalies could be readily detected if they were to occur in offspring resulting from IVF and ET. We have previously reported normal cleavage development of IVF rhesus oocytes up to the morula stage (6). However, conclusive evidence for normal fertilization and embryogenesis can be obtained only from birth of normal offspring after ET. We now report the birth of a healthy rhesus male infant after nonsurgical (intrauterine) transfer of two IVF embryos to a recipient female. This birth provides validation of the IVF procedures used in our laboratory and offers an experimental model for studies on fertilization and embryonic development in primates. MATERIALS AND METHODSAll female monkeys used in this study were housed separately from males. Procedures for recovery of oocytes from gonadotropin-stimulated rhesus monkeys, preparation of spermatozoa, IVF, and embryo culture were all as previously described (6). Five female rhesus monkeys were each given a total of 2150 international units of pregnant mare serum gonadotropin (PMSG) from day 4 of the menstrual cycle (day 1 = first day of menses) through day 15. On day 16, 4000 international units of human chorionic gonadotropin was given, and aspiration of follicles during laparoscopy was performed 30 hr later. Oocytes were incubated individually in drops of culture medium TALP (9). After 5-7 hr, oocyte drops (90 Al) were inseminated with 10 1.l of washed ejaculated ...

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