| Interspecies somatic cell nuclear transfer (iSCNT) provides a potential solution to overcome the problem of oocyte shortage for therapeutic cloning. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF) and intraspecies SCNT. The objectives of this study were to choose effective embryo reconstruction methods and culture media for in vitro production of Cynomolgus monkey-pig iSCNT embryos. The results are as follow:1. In this study, monkey and porcine skin fibroblasts were used as donor cells for reconstruction with porcine enucleated oocytes. The reconstructed embryos were then cultured under PZM-3. No apparent difference was found between monkey-pig (M/P)-iSCNT, and pig-pig (PP)-SCNT embryos in cleavage rate (70.0vs.76.6%, respectively; p>0.05);. However, MP-iSCNT embryos failed to develop beyond the16-cell stages. PCR amplification of monkey-specific and pig-specific microsatellite markers showed that the DNA of monkey iSCNT embryos and pig SCNT embryos were identical to that of their donor. 2. Changes in chromatin configuration of cloned embryos were observed by staining them, atl hr post-fusion, with the DAPI stain. M/P-iSCNT displayed low rate of PCC, and electroporation in Ca2+-free fusion medium had resulted in increased PCC, but no embryo reconstruction method tend to enhance monkey iSCNT embryos development.3. we cultured the enbryos using four different culture systems:porcine zygote medium-3(PZM-3); Hamster Embryo Culture Medium-10(HECM-10); HECM-10+10%FBS. No apparent difference in cleavage rate was found among these four different culture systems. The iSCNT embryos did not develop into blastocysts in any of the media.Results from this study suggest that the porcine oocytes partly supported cell growth, division and nuclear remodeling of monkey-pig iSCNT embryos, but they were unable to support the development of monkey-pig iSCNT embryos to the blastocyst stage. |
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