Somatic cell nuclear transfer (SCNT) was used to generate patient-specific embryonic stem cells (ESCs) from blastocysts cloned by nuclear transfer (ntESCs). In this study, a total of 135 oocytes were obtained from 12 healthy donors (30-35 years). Human oocytes, obtained within 2 h following transvaginal aspiration, were enucleated using a Spindle Imaging System to position the spindle and chromosomes that lay on the metaphase plate, and a Zona Infrared Laser Optical System was used to open a single hole in the zona pellucida at the ~ 2 o'clock position. Human fibroblasts and lymphocytes were used to construct SCNT embryos. Nearly half (26 of 58) of the oocytes were fused after electrofusion and embryo development rates were 96.2% (two-cell, 25 of 26), 92.3% (four-cell, 24 of 26), 61.5% (eight-cell, 16 of 26), 34.6% (16-cell, 9 of 26), 26.9% (morula, 7 of 26), and 19.2% (blastocyst, 5 of 26), respectively, following incubation in improved G-series sequential medium. One cloned blastocyst was used for STR-DNA identification and genetic polymorphism analysis of mtDNA, and STR-DNA analysis of all cloned blastocysts indicated they were derived from SCNT. Quantitative analysis showed that mtDNA of cloned embryos reflected the change tendency of those observed in human IVF embryos. Our research provides an alternative enucleation approach for producing human SCNT-derived blastocysts, and may aid in providing a new method for human therapeutic cloning.

We reconstructed cloned embryos of different genetic diseases by somatic cell nuclear transfer. We want to establish a system for basic researches and clinical therapies of these diseases. At first, we transferred human fetal fibroblast cells into 58 enucleated human oocytes and 21 cloned embryos were reconstructed by electrofusion. After physical and chemical activation, reconstructed embryos developed in vitro 2-cell embryos (20, 95.2%), 4-cell embryos (19, 90.5%), 8-cell embryos (13. 61.9%), 16-cell embryos (7, 33.3%), morula (6, 28.6%), and blastocysts (4, 19.1%), respectively. The STR loci of reconstructed embryos were the same as donor cells.Embryos produced by inter-species nuclear transfer were performed by put human somatic cells into perivitelline space of enucleated rabbit oocytes. After electrofusion, we obtained 210 cloned embryos from 310 reconstructed embryos. Cloned embryos developed in vitro 2-cell embryos (188, 86.6%), 4-cell embryos (188, 86.6%), 8-cell embryos (188. 86.6%), 16-cell embryos (188, 86.6%), morula (141, 65%), and blastocysts (81, 37. 3%), respectively. We selected 18 reconstructed embryos randomly for amplification by human hu-alpgj and rabbit ra-wap probes. Results showed 16 out of 18 reconstructed embryos were hu-alpgj (+) and the other two were hu-alpgj (-), and all the embryos were ra-wap (-). 10 selected embryos were identified for STR-DNA, and the results showed STR loci of those embryos were consistent with donor cells. Furthermore, we obtained reconstructed embryos of different disease (Alzheimer's Disease, Parkinson's Disease, Spinal muscular atrophy, Diabetes, Tumor, etc). STR loci of different disease reconstructed embryos were the same as donor cells.We also designed studies to explore gene expression associated development, DNA methylation, mitochondria, and telomerase activity. has set up effective platform of nuclear transfer and reconstructed human embryos of different diseases. As a chief scientist, he has accomplished 2 "863" projects and took part in "973' project during the recent 5 years. He has published more than 20 papers at domestic and abroad and reported 3 patents. In addition, he also paid attention to human epididymal functional gene research.