摘要
[目的]为了选择适合大黄鱼头肾细胞(LYCK)的转染试剂并优化转染条件,用于开展后期大黄鱼细胞生物学研究。[方法]分别将LipofectamineTM2000、FuGENE HD、X-tremeGENE 9及X-tremeGENE HP与带有加强型绿色荧光蛋白(EGFP)编码基因的质粒pEGFP-N1转染LYCK细胞。[结果]48 h后通过倒置荧光显微镜观察EGFP的表达情况,发现4种转染试剂中X-tremeGENE HP的转染效果(转染效率~1%,细胞生存率>80%)优于其他三种(转染效率<1%,细胞生存率<50%)。在此基础上进一步优化转染条件发现:当DNA质量(μg)和X-tremeGENE HP体积(μL)的比例为1∶7时,转染效率~10%且细胞生存率>60%。转染试剂联用的方法证实,X-tremeGENE HP和LipofectamineTM2000联用转染效率(转染效率~3%)高于其他混合转染组(转染效率<1%)。[结论]综合考虑转染效果、细胞毒性及经济效益,选用X-tremeGENE HP作为转染介质,DNA质量与X-tremeGENE HP体积比例为1∶7时,能够在LYCK细胞中实现最佳转染效果,这一结果为在利用LYCK细胞开展大黄鱼相关免疫基因研究提供了有效的技术手段。
[Objective]To select a suitable transfection reagent and condition for the large yellow croaker head kidney cells(LYCK),which used to research the cell biology of large yellow croaker.[Method]The pEGFP-N1 plasmid expressing enhanced green fluorescent protein(ECFP)was used to transfect LYCK cells by LipofectamineTM 2000,FuGENE HD,X-tremeGENE 9 and X-tremeGENE HP,respectively.[Result]48 hours after transfection,cells were photographed under an inverted fluorescence microscope.Among different transfection reagents,the effect of transfection with X-tremeGENE HP(transfection efficiency-1%,cell viability>80%)was superior to others(transfection efficiency<1%,cell viability<50%).Furthermore,the highest transfection efficiency and cell viability(transfection efficiency-10%,cell viability>60%)for LYCK cells was obtained when the ratio of plasmid DNA(μg)to X-tremeGENE HP(μL)was 1:7.The method of combining transfection reagents confirmed that the transfection efficiency of X-tremeGENE HP and LipofectamineTM 2000 was higher(transfection efficiency-3%)than that of the other combination groups(transfection efficiency<1%).[Conclusion]Comprehensive consideration the amount of transfection reagents,cytotoxicity and economic benefits,this study preliminarily assumed that the transfection of LYCK cells should adopt X-tremeGENE HP.This result provide effective technical means for study of immune genes of large yellow croaker in LYCK cells.
作者
王贤慧
慕鹏飞
敖敬群
WANG Xian-hui;MU Peng-fei;AO Jing-qun(Biomedical Research Institute,Hubei University of Medicine,Shiyan 442000,China;Key Laboratory of Marine Biogenetic Resources,Third Institute of Oceanography,MNR,Xiamen 361005,China;Institute of Oceanology,Fujian Agriculture and Forestry University,Fuzhou 350002,China)
出处
《生物技术》
CAS
2023年第3期287-292,321,共7页
Biotechnology
基金
国家自然科学基金-青年科学基金项目(42106135)。
关键词
大黄鱼头肾细胞
细胞转染
绿色荧光蛋白
瞬时转染
LYCK
cell transfection
emhanced green fluorescence protein
transient transfection
