Effect of Acteoside as a Cell Protector to Produce a Cloned Dog

Ji Hye Lee1☯, Ju Lan Chun1☯, Keun Jung Kim1 , Eun Young Kim1 , Dong-hee Kim1, Bo Myeong Lee1 , Kil Woo Han1 , Kang-Sun Park1 , Kyung-Bon Lee2 , Min Kyu Kim1*

Contact: joanna.jia@wecistanche.com

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acteoside in cistanche has many effects

Table 5. Microsatellite analysis of the cloned dog.

Table 6. mtDNA sequences of the cloned dog.

are improved with the use of donor cells at the G0/G1 stage relative to the use of donor cells at the G2/M stage [24–28], although it has been reported that donor cells arrested at the G2/M stage of the cell cycle can produce viable cloned piglets [44]. The cell cycle stage of the nucleus donor cells plays a crucial role in the reprogramming events that follow SCNT. Nucleus donor cells arrested at G0/G1 stage efficiently initiate the first DNA synthesis after SCNT [28, 29, 45]. To induce cell cycle synchronization, various chemical inhibitors including acteoside have been used to achieve cell cycle synchronization [46, 47]. As a CDK inhibitor, acteoside is often used to bring about cell cycle synchronization at the G0/G1 stage. Lee et al. reported that acteoside hindered the cell cycle progression beyond the G1 phase, thus preventing leukemia cell proliferation. In addition, the level of CDK was reduced but the levels of CDK inhibitors was significantly increased [38].

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The present study compared the effects of acteoside to the other two common cell synchronization methods to investigate the effect of cell synchronization on the efficiency of SCNT. Canine fetal fibroblasts were treated with various concentrations of acteoside, serum starvation, and contact inhibition; the percentage of cells at the G0/G1 stage in the three treatment groups was compared. Serum starvation was found to be the most effective method for cell cycle synchronization at the G0/G1 stage, and there was no significant difference between acteoside and contact inhibition. However, serum starvation-induced a significantly higher level of ROS. Previous studies reported that the increase of ROS damages cell membranes and induces apoptosis thereby diminishing the efficiency of embryo development. Moreover, ROS increases DNA fragmentation that induces the cell block and delays embryo development in humans and pigs [48–51]. Acteoside treatment showed no difference in synchronizing cell cycle at G0/G1 stage compared to contact inhibition. However, acteoside induced significantly less ROS activity compared to the other two cell cycle synchronization methods. In addition, acteoside treatment induced significantly less apoptosis and necrosis than contact inhibition and serum starvation. The result is also congruent with the previous studies that showed the occurrence of more apoptotic events after cell cycle synchronization with serum starvation than with contact inhibition [32, 52]. Concurrent with the reduction in the rate of apoptosis, the acteoside treatment group also showed higher cell survival than the contact inhibition group. Serum starvation resulted in massive cell death compared to both acteoside treatment and contact inhibition.

Nucleus donor cell cycle synchronization at G0/G1 stage is a crucial step in a successful SCNT embryo and ultimately in the production of cloned animals. ROS has been regarded as one of the main causes of cell death and apoptosis during embryo development. In this study, acteoside was investigated to determine whether it would be a useful alternative method for inducing G0/G1 stage cell-cycle synchronization in canine fetal fibroblasts as nuclear donor cells. Induction of cell cycle synchronization by acteoside treatment of nuclear donor cells reduced ROS and apoptosis, which contributed to the improvement of in vitro development of SCNT embryos. Embryos cloned using acteoside-treated donor cells were transferred into surrogate mother dogs and one healthy cloned dog was produced successfully, which did not happen with embryos from the contact inhibition group.

In conclusion, this study demonstrated that acteoside, which is a CDK inhibitor, induces successful cell cycle synchronization of the canine fibroblasts at the G0/G1 stage for use as nuclear donor cells, and also protects them from apoptosis by reducing oxidative stress. The cytoprotective effect of acteoside, combined with the ability for cell cycle synchronization, contributed to improving the in vitro developmental competence of SCNT embryos. Therefore, acteoside would be an effective reagent to enhance the cloning efficiency to produce cloned animals.

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Acknowledgments

The author would like to thank Dr. John Hammond from USDA-ARS for his scientific suggestions and writing support for the manuscript.

Author Contributions

Conceived and designed the experiments: JHL JLC MKK. Performed the experiments: JHL KJK EYK DHK BML KWH KSP. Analyzed the data: JLC KBL. Contributed reagents/materials/ analysis tools: KJK EYK DHK BML KWH KSP. Wrote the paper: JHL JLC. Funding acquisition and supervision: MKK.

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