earticle

영어

Somatic cell nuclear transfer(SCNT) using micromanipulator have been used as a traditional cloning technique and applied various researches during the last decades. However the micromanipulator is expensive and relatively longer training period is required to operate it efficiently. Handmade cloning(HMC) is an alternative cloning method in a simplified way compared to the traditional cloning. Here, we suggested modified porcine handmade cloning(mHMC) as a new approach to clone porcine embryos as a substitute of the traditional SCNT. In mHMC, a nucleus was removed by an aspiration method by using a glass pipette, instead of bisection method. In this study, we investigated the efficiency of the enucleation method in mHMC by assessing the developmental competence of embryos in comparison with the traditional SCNT. The efficiency of enucleation was evaluated based on the rate of the accuracy and oocyte survivability. The accuracy of enucleation was lower in mHMC compared to those in SCNT(98.01±0.57 vs. 83.83±2.47), and the rate of survived oocytes was also lower in mHMC(96.50±0.84 vs. 90.10±2.11, respectively). And the developmental competence was assessed. The blastocyst rate was significantly higher in mHMC group(13.53±2.08 vs. 20.48±0.99). The levels of apoptosis and ROS were investigated to evaluate embryo quality. The expression of ROS and apoptosis-related genes showed no difference between groups. And the relative expressions of mRNA of pluripotency genes and reprogramming genes were evaluated. Although DNMT1 and DNMT3α were not differently expressed in two groups, the expression of the one of pluripotent gene, Oct4 was significantly higher in mHMC. In conclusion, based on the comparable results of mMHC and SCNT, the mHMC could be a suitable alternative technique to clone embryos in cost effective way compared to traditional SCNT.