45 IN VITRO DEVELOPMENT OF BOVINE TRANSGENIC NUCLEAR TRANSFER EMBRYOS IN SERUM-FREE AND SERUM-SUPPLEMENTED MEDIA
J. Y. Lee A , S. G. Lee A , E. J. Jung A , S. H. Jeong A B , C. J. Yang A B , Y. W. Jeong A , S. H. Hyun A B , Y. W. Kim B , T. Shin A , E.-B. Jeung C and W. S. Hwang AA Sooam Biotech Research Foundation, Bangbae 3 Dong, 1027-4 SooAm Building, Seoul 137-851, Republic of Korea;
B Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University,Cheongju 361-763, Republic of Korea;
C Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University,Cheongju 361-763, Republic of Korea
Reproduction, Fertility and Development 23(1) 128-129 https://doi.org/10.1071/RDv23n1Ab45
Published: 7 December 2010
Abstract
Novel serum-free media (IVD101) has been shown to be effective for the production of in vitro-produced embryos for subsequent implantation into cows (Hoshi 2003 Theriogenology 59, 675–685). The objective of the present study was to determine whether serum-free embryo cultivation during preimplantation stage could be used for the production of bovine transgenic nuclear transfer embryos. Somatic cell nuclear transfer (SCNT) embryos were produced by using donor cells containing a vector to induce the production of human erythropoietin in cow's milk. αS1-casein was selected as the promoter to be used in this study through the specific promoter activity test, and enhanced green fluorescent protein(EGFP) gene was attached to the CMV promoter to allow observation of the donor cell during the experiment. Adult fibroblast cells were transfected with lipofectamine. After G418 selection, the transfected cells were injected to the enucleated oocytes, and injected embryos were accomplished by cell-to-cell fusion. These embryos were then activated with calcium ionomycin and 6-dimethylaminopurine. The reconstructed embryos were cultured in IVD101 and mSOF media at 38.5°C, in a 5% CO2, 5% O2, and 90% N2 atmosphere. Embryos were cultured for 4 days, followed by addition of FBS in case of mSOF media. On day-7, the developmental ability and the number of cells in the reconstructed embryos were determined. Statistical analysis of embryo development data was carried out using unpaired t-test, or ANOVA. There were no significant differences in the cleavage rate (69.6 ± 3.2% v. 64.5 ± 5.0%), blastocyst rate (18.7 ± 1.3% v. 22.0 ± 1.6%), and cell number (113.9 ± 7.5 v. 103.6 ± 7.9) between IVD101 and mSOF+FBS cultured embryos. These results indicated that serum-free media did not reduce the developmental competence of SCNT embryos compared with serum-supplemented media. Further studies are required to investigate whether this serum-free transgenic embryo cultivation could be used for developmental potential in terms of full-term development after embryo transfer.
