【摘要】 家禽基因编辑广泛应用于家禽生产实践，如提高家禽生产性能、抗病育种与载体疫苗研发、改善动物福利、改善禽蛋消费的安全性、在禽蛋中生产药用蛋白等。目前，家禽基因编辑仍然依赖传统的体外或体内原始生殖细胞(primordial germ cells, PGC)途径，编辑效率低下。体外直接对精子进行基因编辑，即精子转染辅助的基因编辑(sperm transfection assisted gene editing, STAGE)，有望提高基因编辑效率，而电穿孔是安全、经济和简便的精子转染方法。本研究在不同电穿孔条件下，将Cy-3标记的DNA导入广西麻鸡(Gallus gallus)精子细胞，使用计算机辅助精液分析系统(computer assisted sperm analysis system,CASA)评估精子动力学参数，通过荧光显微镜和流式细胞术评估精子细胞膜和顶体完整性、线粒体膜电位和外源DNA吸纳效率，筛选最佳电穿孔条件。结果显示，在优化的电穿孔条件(100 V, 5 ms, 5次脉冲)下，精子细胞对外源DNA吸纳效率达57.1%，与受精能力相关的精子前进运动性、顶体完整性和线粒体膜电位无明显变化，只有精子总运动性和细胞膜完整性显著下降(P<0.05)，对精子损伤最小。本研究为电穿孔介导的STAGE在家禽基因编辑中的应用提供理论依据。更多还原

【Abstract】 Gene editing in poultry is widely used in poultry industry, such as enhancing economic traits of poultry, breeding for disease resistance and developing vector vaccines, improving poultry welfare, improving the safety of poultry egg consumption, and producing pharmaceutical proteins in eggs, and so on. At present,gene editing in poultry relies on the traditional in vitro or in vivo primordial germ cell(PGC) pathways, and gene editing efficiency is much lower. In vitro gene editing of sperm,or sperm transfection assisted gene editing(STAGE), is a promising approach to improve gene editing efficiency, and electroporation is a safe,economical and simple method for sperm transfection. The purpose of this study was to electroporate Cy-3fluorescein-labeled DNA into Guangxi Partridge chicken(Gallus gallus) sperm cells to screen the optimal electroporation conditions through evaluating sperm dynamics parameters using a computer-assisted sperm analysis system(CASA), investigated sperm cell membrane and acrosome integrity, mitochondrial membrane potential and foreign DNA take-up efficiency by fluorescence microscopy and flow cytometry under different combination of electroporation parameters. The results showed that under the optimized electroporation conditions(100 V, 5 ms, 5 pulses), the DNA take-up efficiency was up to 57.1%, and no significant changes were observed in the fertilization-related parameters such as progressive motility of sperm, sperm acrosome integrity, and sperm mitochondrial membrane potential, significant decrease was seen in total motility of sperm and sperm membrane integrity(P<0.05), showing minimum damage to sperm cells. This study provides a theoretical basis for the application of electroporation mediated STAGE in poultry gene editing.更多还原