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| 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 |
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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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Received: 29 April 2022
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