Two Transfection Reagents of Epithelioma Papulosum Cyprinid Cell Transfection: Condition Optimization and Application
LI Shangqi1,2, XU Ziming3, ZHAO Ran1, SUN Xiaoqing1, ZHANG Yan1, LI Jiongtang1
1. Beijing Key Laboratory of Aquatic Biotechnology, Key Laboratory of Aquatic Genomes, Ministry of Agriculture and Rural Affairs, Biotechnology Research Center, Chinese Academy of Fishery Sciences, Beijing 100141, China; 2. Institute of Fisheries Engineering, Chinese Academy of Fishery Sciences, Beijing 100141, China; 3. National Experimental Teaching and Demonstration Center of Fisheries Science, Shanghai Ocean University, Shanghai 201306, China
Abstract：To solve no data support of reagent dose and sample time in gene overexpression research with epithelioma papulosum cyprinid(EPC) cell transfection, two transfection reagents, Lipofectamine® 2000 and FuGENE® HD, were used to transfect a fluorescent protein tagged plasmids into EPC cells according to varied dose groups with transfection efficiencies recorded at multi-temperature and multiple time points. EPC cells were plated on 35mm cell plate at 28 ℃, with the maximal transfection efficiency (5.92±0.52)% when transfected by 16 μL Lipofectamine®2000 with 4 μg DNA after 48 h post transfection;meanwhile (9.81±0.33)% EPC cells were fluorescence positive by 12 μL FuGENE® HD with 4 μg DNA after 72 h post transfection. To test these optimized conditions and explore the transfection performance differences of the two reagents in transcriptional regulation research, an EGFP tagged overexpression vector was conducted which loaded a common carp transcription factor peroxisome proliferators-activated receptors alpha (PPARα), which was involved in highly unsaturated fatty acid biosynthesis. The transfection efficiency of Lipofectamine® 2000 was found to be 4% and that of FuGENE® HD 8%. Then, real-time quantitative PCR was performed to detect the expression level of candidate target gene fatty acid desaturase 2 (fads2). The candidate target genes were similarly up-regulated by transcription factor overexpression despite transcriptional activation by the lipid from transfection reagent, compared with strict control groups transfected with no-load plasmids. In this study, the dose of two transfection reagents (Lipofectamine® 2000 and FuGENE® HD) to DNA and their sampling time of EPC cell transfection were optimized, and tested with a transcriptional regulation instance of common carp, which contributed to effectively conduct gene research with EPC cells and better promoted breeding innovation of fishery germplasm resource.
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2024, Vol. 43 
