Abstract

There are no reports of cloning by embryo splitting in the cynomolgus monkey, but production of genetically identical monkeys would have tremendous implications for biomedical research, especially for immunological studies, production of disease models, and behavioral science. Cloning would also reduce the number of animals required for the above research by increasing experimental reproducibility. In this study, we tried to produce cynomolgus monkey offspring by embryo splitting and embryo transfer. Controlled ovarian stimulation and oocyte recovery have been previously described by Torii et al. (2000 Primates 41, 39–47). Cumulus-free mature oocytes were fertilized by intracytoplasmic sperm injection. Single spermatozoa were individually immobilized by scoring their tails and picking them up with the injection pipette. The denuded oocyte was held by the holding pipette with the polar body in the 12 o'clock position. The injection pipette was then inserted at the 3 o'clock position and was introduced into the cytoplasm, breaking the ooplasmic membrane by pulling gently. One spermatozoon was injected into the cytoplasm. The injected oocytes were cultured at 38°C in 5% CO₂, 5% O₂ and 90% N₂ in CMRL-1066 medium (Invitrogen, Grand Island, NY, USA) containing 20% calf serum (CS, Invitrogen) for 2–3 days. Splitting was performed using 4- to 7-cell-stage embryos. The zona pellucida was disrupted with acidic Tyrode's solution, and individual blastomeres were separated from the zona-free embryos by 0.25% trypsin-EDTA with added CaCl₂ (<1 min). After transferring the zona-free embryos into TALP-HEPES medium, blastomeres were dissociated by pipetting with a 40–50 µm micropipette 4–5 times. These blastomeres were then transferred into empty zonae that had been produced from immature oocytes by the aspiration of ooplasm with a micromanipulator. Sixteen embryos underwent blastomere separation and a total of 33 split embryos were produced. After being cultured for 2–3 h in CMRL-1066 medium containing 20% CS, 30 of these split embryos, comprising 1–4 blastomeres each, were transferred into the oviducts of 23 fertile surrogate mothers at 0 to 5 days after ovulation. Pregnancy was confirmed in two animals (8.7%; 2/23) by ultrasound approximately 30 days after transfer. The pregnancies were uneventful and two normal healthy babies were born without any assistance 159 days after transfer. The low pregnancy rate could be due to the presence of fewer cells in the smaller split embryos, the ruptured zona pellucida, or the in vitro micromanipulation of embryos during blastomere separation and reconstruction. Here we report the first production of viable cloned offspring produced by blastomere separation in the cynomolgus monkey. Since we have previously succeeded in establishing ES cell lines from isolated blastomeres, in the future we will be able to produce genetically identical monkeys from a single 4- to 8-cell-stage embryo using those ES cell lines and the embryo splitting technique.