Development of a modified method of handmade cloning in dromedary camel

摘要

In this study, a modified method of handmade cloning (m-HMC), which had been originally developed in sheep, was used for somatic cell nuclear transfer (SCNT) in the dromedary camel. The unique feature of m-HMC over current SCNT methods lies in the use of a simple device (a finely drawn micropipette made of Pasteur pipette) for chemically-assisted enucleation of oocytes under a stereomicroscope with improved efficiency and ease of operation. Using this system, the throughput of cloned embryo reconstitution was increased over 2-fold compared to the control SCNT method (c-NT). Stepwise measurement of reactive oxygen species (ROS) revealed that method, steps, and duration of SCNT all influenced oxidative activity of oocytes, but their impact were not similar. Specifically, UV-assisted oocyte enucleation was identified as the major source of ROS production, which explained significantly higher total ROS of reconstituted embryos in c-NT compared to m-HMC. Fusion efficiency (95.3±3.3 vs. 75.4±7.6%) and total efficiency of blastocyst development (22.5±3.0 vs. 14.1±4.3%) were significantly higher in m-HMC compared to c-NT, respectively, and blastocysts of transferable quality were obtained in similar rates (41.9±8.2 vs. 48.0±15.2%, respectively). Significance differences were observed in total cell number (155.3±13.6 vs. 123.6±19.5) and trophectoderm (145±9.5 vs. 114.3±15.2), but not inner cell mass (10.3±4.1 vs. 9.3±5.3) counts between blastocysts developed in c-NT compared to m-HMC, respectively. However, expression of key developmental genes (POU5F1, KLF4, SOX2, MYC, and CDX2) was comparable between blastocysts of both groups. The introduced m-HMC method might be a viable approach for efficient production of dromedary camel clones for research and commercial utilization.