手工克隆小鼠胚胎的研究

与传统克隆相比,手工克隆使用较为简单的仪器设备和技术操作即可生产克隆动物。小鼠作为模式实验动物在多领域得以应用。本研究中采用杂交胎鼠细胞系提供核供体细胞,在含细胞松弛素(Cytochalasin B,CB)和链蛋白酶(Pronase)及2%血清的M199 hepes缓冲液滴(CBMP)中消化透明带并切割去核,电融合后1 h,重构胚置于含Sr Cl2及CB(5μg/m L)的无钙体外培养液(Ca2+-free IVC)中激活6 h,体外培养液(IVC)中清洗3遍后,移入WOW系统培养,培养至小鼠HMC囊胚阶段。研究发现:1)CBMP液中Pronase的浓度相比20μg/m L和100μg/m L,卵母细胞透明带在10μg/m L组需要更长时间消化至消失,较有利于操作;2)直流电(DC)80 V,持续40μs,融合率达60%。3)含10 mmol/L Sr Cl2的化学激活液处理的重构胚与5mmol/L组相比,囊胚更高(29±3%∶13±3%,P0.05),差异显著;4)两种体外培养液,CZB与m PZM,在IVC囊胚率上无显著差异(29±3%∶24±5%,P0.05),而使用CZB可获得较高的囊胚率。本实验首次采用HMC方法体外条件下生产小鼠克隆胚胎,通过摸索透明带消化时间、电激活参数、化学激活试剂及培养液,尝试小鼠体外生产克隆胚胎的新方法,并取得了初步成果,技术优化及效率提高问题仍需要更进一步的研究。

Study on Handmade Cloned Embryos in Mice

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 M199hepes buffer （CBMP）; lhour after electric fusion, the reconstructed embryos were set into Ca^2＋ -free activation medium containing SrCl2 and CB（5 mu g/mL） 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/mL and 100μg/mL, CBMP with 10μg/mL pronase was suitable for operation due to zona pellucid had sufficient time for digestion while enucleating. Secondly, 80v DC, with 401.rs endurance time, the fusion rate achieved 60%. Third, chemical activation with10mM SrCl2 significantly increased the blastocyst rate compared with 5mM （29 ± 3% vs 13 ±3%, P 〈0. 05）. Fourthly, a comparison between CZB and mPZM, 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 and higher efficiency are required further research.