Study on Handmade Cloned Embryos in Mice

作者：Y Chen · 年份：2014 · 研究领域：Pluripotent Stem Cells Research、Animal Genetics and Reproduction、Reproductive Biology and Fertility

Compared with the traditional cloning, cloned animals could be made by more simple equipments and technical operations,and that procedure is handmade cloning( HMC).As model animals,mouse are applied in many fields. In the present study,nuclear donor came from hybrided fetal cell lines; zona pellucida digestion and enucleating happed in droplets of Cytochalasin B( CB) and Pronase and 2% serum M199 hepes buffer( CBMP); 1hour after electric fusion, the reconstructed embryos were set into Ca2 +- free activation medium containing Sr Cl2 and CB( 5 mu g / m L)for the next 6 hours; finally,the embryos were culture to blastocyst stages in a WOW system with culture medium( IVC). The results show that: Firstly,compared with 20μg / m L and 100μg / m L,CBMP with10μg / m L pronase was suitable for operation due to zona pellucid had sufficient time for digestion while enucleating. Secondly,80 v DC, with 40μs endurance time, the fusion rate achieved 60%. Third,chemical activation with10 m M Sr Cl2 significantly increased the blastocyst rate compared with 5m M( 29 ±3% vs 13 ± 3%,P 0. 05). Fourthly,a comparison between CZB and m PZM,there was no significant difference in blastocyst rate( 29 ± 3% vs 24 ± 5%,P 0. 05),but more blastocysts could be obtained when embryos cultured in CZB.After investigate the digestion time,fusion parameters,chemical activation and culture mediums,a novel HMC procedure for cloned embryos of mouse is established for the first time,although the technical optimization ...