摘要
低转染效率一直是利用基因转染技术研究柔嫩艾美耳球虫的主要屏障。为了进一步提高现有的转染效率,本研究分别利用传统的Bio-Rad Gene Pulser电穿孔仪和新型的NucleofectorⅠ核转仪对柔嫩艾美耳球虫子孢子的转染条件进行了优化。通过对外源DNA浓度、电场强度、环境温度及NucleofectorⅠ核转仪的内置程序这些关键参数的探索,最终发现利用Bio-Rad Gene Pulser电穿孔仪设置电压为2000V,电容25μF,利用4mm电击杯进行电转时,转染效率最高,达到3.8×10-4,此时最佳质粒浓度为50μg/107子孢子。
Low transfection efficiency has been the major hurdle for gene transfection in Eimeria tenella.In order to overcome this difficulty,the electroporation parameters were optimized for traditional electroporation apparatus of Bio-Rad Gene Pulser and newly developed Amaxa’s Nucleofector I device,respectively.By adjusting series of key parameters,which include the concentration of plasmid DNA,the electric field intensity,the ambient temperature and bult-in programs of Nucleofector I,we found the optimal condition for electroporation of E.tenellasporozoites is at a voltage of 2000Vand a capacitance of 25μF,using 50μg plasmids/10^7 sporozoites and cuvettes with a 4mm gap,which together resulting in a transfection efficiency of 3.8×10^-4.
出处
《中国畜牧兽医》
CAS
北大核心
2010年第11期53-56,共4页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(30471298
30871862)
关键词
柔嫩艾美耳球虫
基因转染
转染效率
Eimeria tenella
gene transfection
transfection efficiency
