| Autor: |
McLean ZL; Reproduction, AgResearch, Ruakura Research Centre, Hamilton, New Zealand.; Applied Translational Research Group and Centre for Brain Research, School of Biological Sciences, University of Auckland, Auckland, New Zealand., Appleby SJ; Reproduction, AgResearch, Ruakura Research Centre, Hamilton, New Zealand.; Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand., Fermin LM; Reproduction, AgResearch, Ruakura Research Centre, Hamilton, New Zealand., Henderson HV; Knowledge and Analytics, AgResearch, Ruakura Research Centre, Hamilton, New Zealand., Wei J; Reproduction, AgResearch, Ruakura Research Centre, Hamilton, New Zealand., Wells DN; Reproduction, AgResearch, Ruakura Research Centre, Hamilton, New Zealand., Oback B; Reproduction, AgResearch, Ruakura Research Centre, Hamilton, New Zealand.; Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand. |
| Abstrakt: |
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. |
