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Controlled Cytoplast Arrest and Morula Aggregation Enhance Development, Cryoresilience, and In Vivo Survival of Cloned Sheep Embryos
Cellular Reprogramming ( IF 1.6 ) Pub Date : 2021-02-02 , DOI: 10.1089/cell.2020.0078
Zachariah Louis McLean 1, 2 , Sarah Jane Appleby 1, 3 , Lisanne Monique Fermin 1 , Harold Victor Henderson 4 , Jingwei Wei 1 , David Norman Wells 1 , Björn Oback 1, 3
Affiliation  

Zona-free somatic cell transfer (SCT) and embryo aggregation increase throughput and efficiency of cloned embryo and offspring production, respectively, but both approaches have not been widely adopted. Cloning efficiency is further improved by cell cycle coordination between the interphase donor cell and metaphase-arrested recipient cytoplast. This commonly involves inclusion of caffeine and omission of calcium to maintain high mitotic cyclin-dependent kinase activity and low calcium levels, respectively, in the nonactivated cytoplast. The aim of our study was to integrate these various methodological improvements into a single work stream that increases sheep cloning success. We show that omitting calcium during zona-free SCT improved blastocyst development from 6% to 13%, while caffeine treatment reduced spontaneous oocyte activation from 17% to 8%. In a retrospective analysis, morula aggregation produced high morphological quality blastocysts with better in vivo survival to term than nonaggregated controls (15% vs. 9%), particularly after vitrification (14% vs. 0%). By combining cytoplast cell cycle control with zona-free embryo reconstruction and aggregation, this novel SCT protocol maximizes the benefits of vitrification by producing more cryoresilient blastocysts. The presented cloning methodology is relatively easy to operate and further increases throughput and efficiency of cloned embryo and offspring production. Integration of additional reprogramming steps or alternate donor cells is straightforward, providing a flexible workflow that can be adapted to changing experimental requirements.

中文翻译:

受控的细胞质体捕获和桑葚聚集增强克隆羊胚胎的发育、低温弹性和体内存活

Zona-free 体细胞移植 (SCT) 和胚胎聚集分别提高了克隆胚胎和后代生产的吞吐量和效率,但这两种方法都没有被广泛采用。间期供体细胞和中期停滞受体细胞质之间的细胞周期协调进一步提高了克隆效率。这通常涉及在未活化的细胞质中分别加入咖啡因和省略钙以维持高有丝分裂细胞周期蛋白依赖性激酶活性和低钙水平。我们研究的目的是将这些不同的方法改进整合到一个单一的工作流程中,从而提高绵羊克隆的成功率。我们表明,在无透明带 SCT 期间省略钙可以将囊胚发育从 6% 提高到 13%,而咖啡因处理将自发的卵母细胞活化从 17% 降低到 8%。在回顾性分析中,桑葚聚集产生了具有更好形态质量的囊胚。体内存活到足月比非聚集对照(15% 对 9%),特别是在玻璃化后(14% 对 0%)。通过将细胞质细胞周期控制与无带状胚胎重建和聚集相结合,这种新的 SCT 方案通过产生更多的低温弹性囊胚来最大化玻璃化的好处。所提出的克隆方法相对容易操作,并进一步提高了克隆胚胎和后代生产的吞吐量和效率。额外的重编程步骤或替代供体细胞的集成很简单,提供了一个灵活的工作流程,可以适应不断变化的实验要求。
更新日期:2021-02-03
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