76 PREOVULATORY EMBRYO TRANSFER INCREASES SUCCESS OF PORCINE SOMATIC CLONING

Abstract

Somatic cloning has emerged as a valuable tool for the production of transgenic animals. However, the technology still suffers from low efficiencies (<1%). In the present study, we used 2 different recipient synchronization protocols and assessed the effects on the cloning efficiency in pigs using triple transgenic donor cells within a bigger study on xenotransplantation. Double transgenic adult fibroblasts (hCD59/DAF) were transfected with a construct coding for human thrombomodulin (hTM). After selection with 800 µg mL⁻¹ G418 for 14 days, cells were analyzed for integration of the construct by PCR and visually selected for expression of the hTM-GFP fusion protein. One positive cell clone was used for all nuclear transfers. Nuclear transfer (NT) was performed using in vitro-matured oocytes, as previously described (Hoelker et al. 2005 Cloning Stem Cells 7, 35–44). Peri-puberal German Landrace gilts were used as recipients in 2 different synchronization protocols. Group 1 received 1200 IU pregnant mare serum gonadotropin (PMSG) IM 117 h prior to embryo transfer, followed by 500 IU human chorionic gonadotropin (hCG) 72 h later. Nuclear transfer complexes were transferred surgically 46 h later, approximately 6 h post-ovulation. Group 2 was synchronized by administration of 5 mL Altrenogest for 12 days (20 mg gilt/day). At the end of treatment, the animals received 1000 IU PMSG IM, followed by an injection of 500 IU hCG 80 h later. Cloned embryos were transferred surgically approximately 20 h prior to ovulation. Maintenance of pregnancy was supported by injections of 1000 IU PMSG on Day 11 and 500 IU hCG on Day 14 of gestation. Animals were checked for pregnancy by ultrasound 25 days after transfer (Table 1). In group 1, only one pregnancy could be maintained, leading to the birth of one piglet after caesarean section. In contrast, 12 pre-ovulatory transfers resulted in 9 pregnancies with a total of 47 cloned piglets born. All offspring had normal birth weights (1.0–1.5 kg) and showed no malformations. Average litter size in group 2 was 5.2, and the overall efficiency was 3.1% and 4.4% when related to pregnant recipients. The results show that the transfer of NT-complexes into oviducts of pre-ovulatory recipients significantly improved the success rates of porcine cloning. This is an important feature for the production of transgenic pigs for biomedical and agricultural application.

Table 1.   Results of porcine nuclear transfer

This project is funded by the Deutsche Forschungsgemeinschaft (FOR 535).