202 KNOCK-DOWN AND DEVELOPMENTAL EFFICIENCY OF Arhgap15 GENE KNOCK-DOWN EMBRYOS

Abstract

The technique of RNA interference (RNAi) knock-down is a powerful tool for the analysis of gene function in mammalian cells and eggs. Rho GTPase-activating protein 15 (Arhgap15) is closely related to anaemia and immunity, especially as caused by trypanosome. This study was performed to investigate the effect of Arhgap15 gene knock-down on the developmental competence of bovine embryos in vitro. Bovine fibroblast cells were treated with 50 and 100 nM concentrations of Arhgap15 RNAi, respectively. After 24 h of transfection, the control group showed no change in Arhgap15 mRNA level, whereas mRNA expression in the RNAi-treated donor cells was obviously decreased in a dose-dependent manner. These RNAi-treated cells were then transferred into enucleated bovine oocytes to analyse the consequence of RNAi-mediated Arhgap15 gene knock-down. In the control group, cleavage and blastocyst development rates were 75% (102/136) and 14.7% (20/136), whereas those in the knock-down embryos were 81.6% (120/147) and 24.5% (36/147), respectively. The occurrence of cell death by apoptosis was examined in Day 7 blastocysts. Apoptotic cells numbered 12 ± 3.2 in control embryos and 8.9 ± 4.8 in knock-down embryos. Therefore, it can be concluded that RNAi-mediated Arhgap15 gene knock-down in somatic cells did not affect the developmental competence of bovine cloned embryos. This work was supported by grant 120080401034062 from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.