374 PRODUCTION OF TRANSGENIC CLONED PIGLETS BY USING PORCINE FETAL FIBROBLASTS OVEREXPRESSING ENDOTHELIAL NITRIC OXIDE SYNTHASE

Abstract

Vascular function, vascular structure, and homeostasis are thought to be regulated in part by endothelial nitric oxide synthase (eNOS). In addition, eNOS plays an important role in modulating metabolism of skeletal and cardiac muscle in health and disease. The pig is an optimal model for human diseases because of the large number of important similarities between the genomic, metabolism, and cardiovascular systems of pigs and humans. The main goal of this study was to produce pigs that carry an endogenous eNOS gene driven by a Tie-2 promoter and tagged with a V5 His tag so that a better understanding of cardiovascular regulation by eNOS can be gained. Nuclear transfer was conducted to create these animals, and the effects of two different oocyte activation treatments and two different culture systems were examined. Mature oocytes were purchased from BoMed, Inc. (Maidson, WI, USA) and enucleated by micromanipulation. Donor cells were injected into the perivitelline space and electrically fused to the recipient oocyte. Treatment 1 consisted of electrical fusion/activation in 1 mM calcium in mannitol; Treatment 2 consisted of electrical fusion in 0.1 mM calcium in mannitol, followed by chemical activation in 200 µM thimerosal for 10 min followed by 8 mM DTT for 30 min. The fusion rate (71% vs. 82%) was lower (P < 0.0001) in Treatment 2 than in Treatment 1. For those oocytes that fused, the cleavage rates (5.9% vs. 0.4%) at 14-18 h were higher (P < 0.0001) for Treatment 2. Fused oocytes were cultured for 12-18 h in 5% O₂, 5% CO₂, 90% N₂ (low oxygen) or 5% CO₂ in air (normal oxygen), and were then surgically transferred to the oviducts of gilts that exhibited estrus on the day of fusion or the day of transfer. In February four cloned transgenic piglets were born (birth weights were 495, 595, 965, and 685 g). Two were from Treatment 1 and low oxygen, and the other two were from Treatment 2 and normal oxygen, and all are currently healthy. PCR, RT-PCR, and Western blotting analysis confirmed that the pigs were transgenic, made message, and made the fusion protein. Immunohistochemistry confirmed that the fusion protein localized in the endothelial cells of the placental vasculature from the conceptuses as did the endogenous eNOS. In addition to the transgenic males, four wild-type females were born (birth weights were 585 (this one died on Day 4) 445, 930, and 585 g). All four were from Treatment 2 and low oxygen, and three are healthy. In conclusion, viable piglets were produced from both Treatments 1 and 2, as well as in both high and low oxygen conditions. Thus, both activation conditions and culture systems are compatible with development to term. These pigs will serve as the founders for a colony of miniature pigs that will help to elucidate the function of eNOS in regulating muscle metabolism and the cardiorespiratory system.

This work was funded by a grant from the NIH RR18276 and Food for the 21st Century.