Hum. Reprod. Advance Access originally published online on June 22, 2006
Human Reproduction 2006 21(10):2564-2571; doi:10.1093/humrep/del216
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A comparative approach to somatic cell nuclear transfer in the rhesus monkey
1 State Key Lab of Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, Beijing 2 Kunming Primate Research Center and Kunming Institute of Zoology, The Chinese Academy of Sciences, Yunnan, China 3 Institute for Zoo and Wildlife Research (IZW), Berlin, Germany and 4 Oregon National Primate Research Center, Beaverton, OR, USA
5 To whom correspondence should be addressed at: Kunming Primate Research Center and Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming, Yunnan 650223, China. E-mail: wji{at}mail.kiz.ac.cn
6 To whom correspondence should be addressed at: Oregon National Primate Research Center, Beaverton, OR 97006, USA. E-mail: wolfd{at}ohsu.edu
7 To whom correspondence should be addressed at: State Key Lab of Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, Beijing 100860, China. E-mail: qzhou{at}ioz.ac.cn
* The same contribution as first author.
BACKGROUND: Despite the potential utility of primate somatic cell nuclear transfer (SCNT) to biomedical research and to the production of autologous embryonic stem (ES) cells for cell- or tissue-based therapy, a reliable method for SCNT is not yet available. Employing the rhesus monkey as a clinically relevant animal model, we have compared a conventional electrofusion method for SCNT with a one-step micromanipulation (OSM) method. METHODS: A prospective, randomized trial was conducted using only oocytes that were mature [metaphase II (MII)] at collection and a fibroblast-like cell line as nuclear donor cells (fetal fibroblasts). The embryos produced were characterized for in vitro developmental potential, cell number, karyotype and expression of nuclear mitotic apparatus (NuMA) and OCT-4. RESULTS: An in vitro blastocyst development rate of 24.4% was achieved with the OSM method, significantly higher than the 12.2% obtained following electrofusion. SCNT-produced embryos expressed normal karyotypes, cell numbers and NuMA and OCT-4 proteins in most cases. SCNT with male nuclear donor cells resulted in the production of male, SCNT blastocysts, eliminating the possibility of a parthenogenetic origin. Of the four fibroblast cell lines tested as nuclear donor cells, two supported the routine production of blastocysts following SCNT. CONCLUSIONS: The application of a modified SCNT technique (OSM) followed by embryo culture in hamster embryo culture medium-10 (HECM-10) allows, for the first time, the routine production of SCNT blastocysts, most of which appear normal by immunochemical, cytochemical and in vitro developmental criteria. These embryos will provide a resource for isolating ES cells and for studies of nuclear reprogramming by monkey cytoplasts.
Key words: rhesus monkey/somatic cell nuclei transfer/therapeutic cloning
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