52 IMPLANTATION OF TRANSGENIC BOVINE CLONED EMBRYOS DERIVED FROM TRANSFECTED CELLS BY PiggyBac TRANSPOSITION
W. J. Choi A , S. J. Lee B , W. W. Lee A B , S. J. Kim A , I. M. Saadeldin A , J. K. Cho C , B. C. Lee A and G. Jang AA Department of Theriogenology and Biotechnology, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, Republic of Korea;
B Embryo Research Center in Seoul Milk Coop., Gyeonggi-do, Republic of Korea;
C Department of Theriogenology, College of Veterinary Medicine, Chungnam National University, Daejeon, Republic of Korea
Reproduction, Fertility and Development 25(1) 173-174 https://doi.org/10.1071/RDv25n1Ab52
Published: 4 December 2012
Abstract
A gene-delivery system, PiggyBac (PB) transposition, has been applied to transgene expression in mammalian cells or animals. In this study, to produce transgenic cattle, we used PB in bovine fibroblasts and then the transfected cells were microinjected into enucleated bovine oocytes to produce embryos and offspring. For this study, 2 different fluorescence genes (GFP, transcribed by constitutive promoter and RFP, transcribed by tetracycline-dependent promoter), which were flanked by PB sequences, were transfected into the bovine fetal fibroblasts by the FuGENE transfection protocol. The developmental rate of blastocysts among the cleaved embryos derived from GFP cells and doxycycline-induced RFP cells was developed at 23.1% (31/134) and 40.9% (442/1082), respectively. After transferring the GFP- or RFP-expressing blastocysts into recipient cows, pregnancies were detected by ultrasonography from both recipients of GFP or RFP. To know gene expression in fetal stage, embryonic sacs were collected surgically. The primary cells were successfully isolated from both embryonic sacs. Every cell from the GFP embryonic sac expressed GFP. When the cells from the RFP embryonic sac were treated with doxycycline, RFP was homogenously expressed. In conclusion, this study demonstrated that PB transposition could be applied to deliver genes into bovine somatic cells. Furthermore, transgenic embryos from transfected cells using the PB system were developed into blastocysts, implanted, and were able to form embryonic sacs. The PB system will be a useful method to produce transgenic cattle.
This study was financially supported by IPET (grant no. 109023-05-3-CG000, 111078-03-1-CG000) and the BK21 Program for Veterinary Science.
