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The Exploration of Porcine Embryo Knockout System VIA ZFN Technology

Author: ZhangQingXiao
Tutor: CaoShaoXian
School: Nanjing Agricultural College
Course: Animal Genetic Breeding and Reproduction
Keywords: Porcine Reproductive and Respiratory Syndrome Sialoadhesin Zincfinger nuclease electroporation nuclear transfer single cell culture
CLC: S814.8
Type: Master's thesis
Year: 2012
Downloads: 12
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Abstract


The ZFN technology got a rapidly development because of its broad applicability, high-efficiency and high specificity.Zinc-finger nucleases have been successfully used in a variety of cell types and organisms as a rational genome engineering tool. ZFNs consist of a non-specific Fokl endonuclease domain and a specific DNA-binding domain. Zinc-finger nucleases can deliver a targeted genomic double-strand break (DSB) and stimulate gene targeting by several orders of magnitude through activation of cellular DNA repair pathways. It either stimulates local homologous recombination with investigator-provided donor DNA or induces gene mutations at the site of cleavage in the absence of a donor by nonhomologous end joining. This study aims to explore PRRSV resistance breeding by this technology. PRRSV mainly infects porcine alveolar macrophages. And only Sn is sufficient for both PRRSV attachment and internalization. If the Sn gene function missing, that may block the infection of PRRSV virus in pigs. Firstly we have successfully isolated porcine fetal fibroblast cell. And with this cell line, we acquired an optimal electroporation procedure. Meanwhile, in order to produce transgenetic embryo via nuclear transfer smoothly for the follow-up work, we made a lot of optimization on porcine oocytes development in vitro and nuclear transfer system. Finally, this experiment identified ten ZFN plasmids whether they were effective, and established several experimental methods. These methods laid a foundation for the follow-up work.1. Separating pig fetal fibroblast cells (pffcs) and optimizing the transfection system of electroporation. Firstly, we have successfully isolated porcine fetal fibroblast cell. To support the porcine gene function study and application as a basic technology, we explored a high efficiency electroporation protocol for pig fetal fibroblast cells (pFFCs). Firstly, we chose the enhanced green fluorescent protein (EGFP) gene as a report gene. Three factors were optimized, including plasmid concentration, electroporation voltage and pulse duration. Then we carried two different temperature treatments on cells, and made a research on the relation between electroporation voltage and cell apoptosis rate. The result showed that the plasmid concentration is the most important factor among these factors; also the plasmid concentration and electroporation voltage had significant effects on transfected efficiency. The pulse duration and different temperature treatments had no significant different effect on it. In addition, the electroporation voltage had a significant effect on the cell apoptosis rate. In conclusion, we got an optimized protocol that the plasmid final concentration was40μg/ml, the electroporation voltage was280V, and the pulse duration was800μs at room temperature, the highest transfected efficiency was89.8%.2. The optimization of porcine oocytes in vitro development and nuclear transfer system. This study started with porcine oocytes development in vitro and embryo activation in vitro.By optimizing the hormone adding way in mature medium and embryonic activation method, we build a relatively perfect porcine oocytes development method and parthenogenetic development method. Based on this, the rate of porcine oocytes development in vitro was up to80%from the slaughterhouse ovary, and cleavage rate to90%, parthenogenetic blastocyst rate to50%. When applying this method to the somatic nuclear embryos development in vitro for transgenic reserch, we got a blastocyst rate of18%.3. A preliminary exploration on the production of Sn-knockout embryo via ZFN technology. We carried out the transfection in pig fetal fibroblast cells with some ZFN plasmids based on the high-efficiency electroporation method of the third chapter, and extracted the cells’DNA to identify whether the gene of Sn was knockout. Then based on the optimized embryonic development system of the fourth chapter and the transfected pig fetal fibroblast cells to produce nuclear transfered embryos. This paper identified ten ZFN plasmids and all were useless. However, this study established a method that culture single cell to cell strain, so as to select the transfected cell; a rapid detection method of the gene mutation and a single embryonic detection method via PCR.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > General Animal Science > Livestock and poultry breeding > Reproductive biotechnology
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