Transgenic pigs are currently believed to be an important model for biomedical research, including for disease models, pharmaceutical toxicity testing, and regenerative medicine. However, production efficiency of animal disease models using somatic cell NT (SCNT) is very low. One of the main reasons is probably characteristics of the transgene. In this study, we introduce SV40LT oncogene into the fibroblast cells in order to establish immortalized transgenic cell line for producing the pig model of human brain cancer. We evaluated the effect of SV40LT oncogene on transgenic SCNT embryo development. As a results, the cleavage rates (73.8 ± 4.0 and 48.6 ± 2.4 in the normal and SV40LT group, respectively; P < 0.05) and blastocyst formation rates (19.5 ± 1.2 and 5.6 ± 1.8 in the normal and SV40LT group, respectively; P < 0.05) of transgenic SCNT embryos was significantly lower than the case of using normal cells. In addition, we evaluated the transgenic SCNT embryo development of the donor cell transfected with SV40LT and HrasV12 genes (SV40LT+HrasV12 group). As a results, there was no significant difference between the groups in the cleavage rates, but blastocyst formation rates of transfected SCNT embryos (SV40LT+HrasV12 group) was significantly lower than the case of using normal cells (19.5 ± 1.2 and 6.2 ± 1.8 in the normal and SV40LT+HrasV12 group, respectively; P < 0.05). Genes SV40LT or HrasV12 showed a negative effect on SCNT cloned embryo development. Therefore, a Cre/loxP inducible system was applied to producing donor cells transfected with EGFRvIII and SV40LT gene. As a result, the cleavage rates (73.8 ± 4.0 and 68.6 ± 6.6 in the normal and Cre/loxP-EGFRvIII-SV40LT group, respectively; P < 0.05) and blastocyst formation rate (19.5 ± 1.2 and 23.0 ± 3.7 in the normal and Cre/loxP-EGFRvIII-SV40LT group, respectively; P < 0.05) were improved to the same level, when used as a donor cell to a normal cell. In conclusion, these results indicated that harmful effects of transgenic SCNT embryo development caused by the characteristics of the inserted genes can be overcome through the inducible system. Further studies are needed to experiment with mRNA expression of apoptotic gene and target gene in 4- to 8-cell embryos and blastocysts.
This work was supported, in part, by a grant from the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ011077, PJ011288), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean government (NRF-2013R1A2A2A04008751), Republic of Korea.
Establishment of a transgenic cell line stably expressing human cytochrome P450 2C18 and identification of aCYP2C18clone with exon 5 missing