Application of the modified handmade cloning technique to pigs

Abstract

Although somatic cell nuclear transfer (SCNT) is frequently employed to produce cloned animals in laboratories, this technique is expensive and inefficient. Therefore, the handmade cloning (HMC) technique has been suggested to simplify and advance the cloning process, however, HMC wastes many oocytes and leads to mitochondrial heteroplasmy. To solve these problems, we propose a modified handmade cloning (mHMC) technique that uses simple laboratory equipment, i.e., a Pasteur pipette and an alcohol lamp, applying it to porcine embryo cloning. To validate the application of mHMC to pig cloning, embryos produced through SCNT and mHMC are compared using multiple methods, such as enucleation efficiency, oxidative stress, embryo developmental competence, and gene expression. The results show no significant differences between techniques except in the enucleation efficiency. The 8-cell and 16-cell embryo developmental competence and Oct4 expression levels exhibit significant differences. However, the blastocyst rate is not significantly different between mHMC and SCNT. This study verifies that cloned embryos derived from the two techniques exhibit similar generation and developmental competence. Thus, we suggest that mHMC could replace SCNT for simpler and cheaper porcine cloning.

Handmade cloning: a developing alternative to traditional SCNT cloning methods

“…The results demonstrate no significant difference between the techniques except in the enucleation efficiency. Enucleation accuracy and oocyte survival rates were higher in the SCNT group than in the m-HMC group (98.01% vs. 83.83%; 96.5% vs. 90%) [17]. However, given the lower costs and relatively simple processes, the report evaluated the m-HMC technique as preferable to the SCNT technique.…”

“…In one of these studies regarding pig cloning, m-HMC and the traditional SCNT cloning techniques were made parallel and analyzed for efficiency. Although the handmade cloning technique had a more straightforward cloning process, it wastes many oocytes which leads to mitochondrial heterosplasmy [17]. Hence, the group developed a m-HMC using Pasteur Pipettes and alcohol lamps.…”

Strategies to Improve the Efficiency of Somatic Cell Nuclear Transfer

“…Therefore, only one oocyte is required to produce a cloned embryo. mHMC has been used to produce cloned blastocysts in sheep [45], pigs [47], and cloned offspring in goats [48], cattle [49], and camels [50]. The use of different SCNT techniques among different cloning laboratories varies due to animal species, equipment availability, and the technical skills of workers.…”