农业生物技术学报2024，Vol.32Issue(12)：2904-2914,11.DOI:10.3969/j.issn.1674-7968.2024.12.019
广西麻鸡精子电穿孔条件的优化
Optimization of Electroporation Conditions for Guangxi Partridge Chicken(Gallus gallus)Sperm
摘要
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-3 fluorescein-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.关键词
鸡/精子电穿孔/精子转染介导的基因转移(SMGT)/DNA吸纳效率Key words
Chicken/Sperm electroporation/Sperm-mediated gene transfer(SMGT)/DNA take-up efficiency分类
农业科技引用本文复制引用
王博永,陈晨,梁谦学,李恭贺,吴文德,郑喜邦..广西麻鸡精子电穿孔条件的优化[J].农业生物技术学报,2024,32(12):2904-2914,11.基金项目
国家自然科学基金(32060738) （32060738）
