Transgenesis in swine
Abstract
Transgenic pigs can serve as valuable models for studying human diseases and have the potential to increase the productivity of animal agriculture. The generation of transgenic pigs with specific genetic modification is a complex procedure that involves numerous steps; in this study various aspects of the technology were investigated in an attempt to make transgenic pig production more effective. Gene targeting in pigs fetal fibroblast cells is extremely challenging. To increase the efficiency, the effects of cell cycle synchronization at the S phase and the inclusion of a nuclear localization signal (NLS) in the targeting vector were examined using the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene as a model. The results indicated that synchronizing the cell cycle at the S phase significantly increased the frequency of random integration of the targeting vector in the genome; at the same time it did not improve gene targeting. In addition, the presence of the NLS in the targeting vector was without any effect under the current experimental conditions. Next, zinc finger nucleases (ZFNs) have been produced to enhance efficiency when targeting the HPRT and adiponectin genes in Ossabaw pig cells. An in vitro assay using the designed HPRT ZFNs showed their potential to induce a double-strand break at a specific site within an HPRT gene sequence. However, no successful targeting was registered when the ZFNs were introduced into fetal fibroblast cells. The results were similar in gene targeting in embryos: no targeted modification was detected. In the process of transgenic pig production, the genetically modified cells are transferred into enucleated oocytes and the reconstructed oocytes are then parthenogenetically activated to initiate embryo development. In the next set of experiments the role of the STIM1 protein, a key molecule involved in Ca2+ signaling was investigated. The results show that STIM1 is essential for the regulation of the sperm-induced Ca 2+ oscillation and subsequent embryo development. These findings suggest that STIM1 is required for normal fertilization in pigs and has a central role in the regulation and maintenance of the sperm-induced Ca2+ oscillation. The final step in the process of transgenic animal production is the culture of the produced embryos until they reach a stage when they are transferred into a recipient female. To develop methods to increase in vitro embryo production, the effects of resveratrol on pig embryo development was studied. At a concentration of 0.5 μM, resveratrol significantly increased the percentage of parthenogenetic embryos that reached the blastocyst stage by day 7. Resveratrol also induced an increase in the total cell number and reduced the expression of the apoptosis-related genes in the blastocysts. The findings will contribute to a better understanding of the mechanisms involved in the generation of transgenic pigs. They may also provide the foundation for the development of new methods to improve the production of swine with targeted genetic modifications.
Degree
Ph.D.
Advisors
Machaty, Purdue University.
Subject Area
Animal sciences
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