50 EFFECTS OF REPROGRAMMING TRANSCRIPTION FACTORS AND CHEMICAL INHIBITORS ON THE DEVELOPMENT OF BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS
B.-C. Yang A , J.-H. An A , H. K. Chung A , S.-J. Jo A , H.-J. Chung A , K.-W. Kim A , D.-W. Han A , S.-S. Lee A , J.-K. Yoo A and J.-K. Park ANational Institute of Animal Science, RDA, Suwon, Republic of Korea
Reproduction, Fertility and Development 25(1) 172-173 https://doi.org/10.1071/RDv25n1Ab50
Published: 4 December 2012
Abstract
Currently, somatic cell nuclear transfer (SCNT) is regarded as the most efficient technique to produce transgenic cattle. However, compared with other techniques, such as IVF and in vivo embryo, the birth rate of SCNT embryos has been disappointingly low. Because incomplete epigenetic reprogramming in SCNT embryos has been suggested as a major reason for this low birth rate, we attempted to improve the success rate by using several lines of donor cells that were reprogrammed by gene transfection and an inhibitor treatment. First, the bovine ear fibroblast cells were transfected with vectors (pCX-OKS-2A and pCX-Myc) carrying 4 genes (Oct-4, Klf4, Sox2, and c-Myc; OKSM). Subsequently, transfected cells were incubated in the presence of one of the following chemical inhibitors: (1) trichostatin A (TSA, 20 nM), (2) 5-aza-20-deoxycytidine (5-aza-dC, 10 µM), or (3) GSK-3/MEK inhibitor (2i; GSK-3, 3 µM and MEK, 1 µM) for 7 days. Throughout the incubation period, cells in each treatment group were analyzed for their histone acetylation status. Subsequently, transfected cells were used as nucleus donors in SCNT and their development rates to the blastocyst stage were estimated. In the present study, it was found that the histone acetylation (H3K9/K14) levels were generally higher in treated groups compared with nontreated control groups. Regarding blastocyst development rate, the OKSM/5-aza-dC (10/49, 20.1 ± 5.9%) combination was better than the other groups: OKSM/TSA (8/77, 11.1 ± 6.9%), OKSM/2i (8/84, 9.5 ± 1.5%), OKSM (6/50, 12.6 ± 6.5%), and negative control group (9/94, 9.6 ± 3.8%; P < 0.05). Embryo development rates appeared to be higher in OKSM/inhibitor-treated groups, although the rate was significantly lower (P < 0.05) in OKSM/2i. In conclusion, our data suggest that transfection with 4 genes and incubating with 5-aza-dC can significantly improve the development rate of bovine SCNT embryos.
