Production of endothelial nitric oxide synthase (eNOS) over-expressing piglets

Abstract

Vascular function, vascular structure, and homeostasis are thought to be regulated in part by nitric oxide (NO) released by endothelial cell nitric oxide synthase (eNOS), and NO released by 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, metabolic and cardiovascular systems of pigs and humans. To gain a better understanding of cardiovascular regulation by eNOS we produced pigs carrying an endogenous eNOS gene driven by a Tie-2 promoter and tagged with a V5 His tag. Nuclear transfer was conducted to create these animals and the effects of two different oocyte activation treatments and two different culture systems were examined. Donor cells were electrically fused to the recipient oocytes. Electrical fusion/activation (1 mM calcium in mannitol: Treatment 1) and electrical fusion (0.1 mM calcium in mannitol)/chemical activation (200 microM Thimerosal for 10 min followed by 8 mM DTT for 30 min: Treatment 2) were used. Embryos were surgically transferred to the oviducts of gilts that exhibited estrus on the day of fusion or the day of transfer. Two cloned transgenic piglets were born from Treatment 1 and low oxygen, and another two from Treatment 2 and normal oxygen. PCR, RT-PCR, Western blotting and immunohistochemistry confirmed that the pigs were transgenic, made message, made the fusion protein and that the fusion protein localized to the endothelial cells of placental vasculature from the conceptuses as did the endogenous eNOS. 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.

Histomorphology of the porcine oviduct

“…It has been reported that NO has a regulatory role in several processes, including ovulation, regression of the corpus luteum, and early embryonic development (8). In the present study, while eNOS expression was not observed in the epithelium of either the ampulla or the fimbria during both the oestrus and luteal phases, in the oestrus phase, strong eNOS expression was determined in both the epithelial and endothelial cells of the isthmus.…”

“…It has been indicated that NO plays a regulatory role in multiple processes, including the secretion of gonadotropins, ovulation, regression of the corpus luteum, and early embryonic development. Furthermore, it has been stated that NO produced by blood vessel endothelium and oviduct epithelium has a regulatory role in the production of the oviduct fluid (8).…”

“…Apart from the many genomic and metabolic similarities between human and pig, the very high level of compatibility observed between the cardiovascular systems of the two species has resulted in the use of pigs as an optimal model in research on human diseases (8). The present study was aimed at the light and electron microscopic examination of the epithelium of the porcine oviduct, the determination of the histochemical structure of the mucus content of the oviduct, and the demonstration of the localisation and distribution of eNOS activity in the porcine oviduct.…”

Production of Transgenic and Non-Transgenic Clones in Miniature Pigs by Somatic Cell Nuclear Transfer

“…As far as the use of pigs as experimental animals is concerned, miniature pigs are smaller and easier to handle than common domestic pigs, and they have been used in a variety of fields, such as medical and pharmacological research [10,[18][19][20][21][22][23][24][25]. However, compared with common domestic pigs, far less information regarding the production of cloned and genetically modified pigs is available for miniature pigs [10,13,14,[26][27][28][29][30][31].In the present study, we conducted a series of experiments including (i) validation of an estrus synchronization procedure for miniature pig recipients, (ii) examination of the developmental competence of nuclear transfer embryos reconstructed with various nuclear donor cells and (iii) comparison of miniature pigs and common domestic pigs as recipients. The results indicate that it is possible to produce cloned miniature pigs using common domestic pigs as the recipients and to produce genetically modified-cloned miniature pigs with a level of efficiency comparable to that for conventionally cloned miniature pigs.…”

Production of Cloned Pigs by Nuclear Transfer of Preadipocytes Following Cell Cycle Synchronization by Differentiation Induction

“…Already, α1,3-galactosyltransferase gene knockout pigs have been produced as organ donors for xenotransplantation [1][2][3][4][5][6][7][8]. In addition, transgenic pigs serving as human disease models, such as those carrying the gene for endothelial cell nitric oxide synthase (eNOS), have been developed [9]. To further advance the use of cloned and genetically modified pigs in research, however, a SCNT system that enables more efficient generation of embryos is needed.…”