电转方法用于modRNA转染细胞的初步研究
发布时间：2021-12-22 21:13
目的探讨电转方法用于modRNA转染细胞的可行性,并筛选出不同细胞电转所需的最适电压和脉冲时间。方法 （1）筛选电转EGFP modRNA进入人成纤维细胞的最佳电压和脉冲时间参数;（2）在此基础上将人成纤维细胞转染n GFP/EGFP+mCherry modRNA;（3）将Hela、293T、3T3三种常见贴壁细胞电转EGFP modRNA。结果 （1）人成纤维细胞电转modRNA的最适电压为440 V,最适脉冲时间为30 ms;（2）nGFP可在核内表达,EGFP和mCherry两种蛋白可同时在胞质中表达;（3）Hela、293T、3T3细胞电转最适电压分别为425 V、400 V和440 V,最适脉冲时间均为30 ms。结论本实验证明（1）电转法转染modRNA进入细胞质和细胞核的可行性;（2）可同时将两种modRNA导入同一个细胞内并表达;（3）最适电压和最适脉冲时间需根据不同的细胞种类分别筛选。
【文章来源】：组织工程与重建外科杂志. 2019,15(04)
【文章页数】：7 页
【部分图文】：
流式检测表达效率和荧光强度Fig.4Detectionofexpressioneffi-ciencyandfluorescenceintensitybyflowcytometry
化图A:12hoursafterelectroporation;B:1dayafterelectroporation;C:2daysafterelec-troporation;D:3daysafterelectroporation;E:4daysafterelectroporation;F:5daysafterelectroporation;G:6daysafterelec-troporation;H:7daysafterelectropora-tion;I:ChangesofexpressionefficiencyofEGFPmodRNAinhumanfibroblasts;J:ChangesoffluorescenceintensityofEGFPmodRNAinhumanfibroblasts图4流式检测表达效率和荧光强度Fig.4Detectionofexpressioneffi-ciencyandfluorescenceintensitybyflowcytometryA：光镜图；B：nGFP荧光图；C：DAPI染色；D：Merge图A:Underlightmicroscopy;B:nGFPfluorescentstaining;C:DAPIstaining;D:Mergediagram图5电转nGFPmodRNA后荧光图Fig.5FluorescencediagramafterelectroporationofnGFPmodRNAA：mCherry荧光图；B：EGFP荧光图；C：DAPI染色；D：Merge图A:mCherryfluorescentstaining;B:EGFPfluorescentstaining;C:DAPIstaining;D:Mergediagram图6电转EGFP+mCherrymodRNA后荧光图Fig.6FluorescencediagramafterelectroporationofEGFP+mCherrymodRNAA：EGFP荧光图；B：DAPI染色；C：Merge图；D：流式分析A:EGFPfluorescentstaining;B:DAPIstaining;C:Mergediagram;D:Flowanalysis图7Hela细胞电转EGFPmodRNA后荧光图Fig.7FluorescenceofHelacellsafterelectroporationwithEGFPmodRNA·219·
iRA,deBizemontT,etal.Oculargenetherapy:areviewofnonviralstrategies[J].MolVis,2006,12:1334-1347.[2]PahleJ,WaltherW.Vectorsandstrategiesfornonviralcancergenetherapy[J].ExpertOpinBiolTher,2016,16(4):443-461.[3]TurnbullIC,EltoukhyAA,FishKM,etal.MyocardialdeliveryoflipidoidnanoparticlecarryingmodRNAinducesrapidandtransientexpression[J].MolTher,2016,24(1):66-75.[4]WolffJA,MaloneRW,WilliamsP,etal.Directgenetransferintomousemuscleinvivo[J].Science,1990,247(4949):1465-1468.A：EGFP荧光图；B：DAPI染色；C：Merge图；D：流式分析A:EGFPfluorescentstaining;B:DAPIstaining;C:Mergediagram;D:Flowanalysis图8293T细胞电转EGFPmodRNA后荧光图Fig.8Fluorescenceof293TcellsafterelectroporationwithEGFPmodRNAA：EGFP荧光图；B：DAPI染色；C：Merge图；D：流式分析A:EGFPfluorescentstaining;B:DAPIstaining;C:Mergediagram;D:Flowanalysis图93T3细胞电转EGFPmodRNA后荧光图Fig.9Fluorescenceof3T3cellsafterelectroporationwithEGFPmodRNA·220·
本文编号：3547101
【文章来源】：组织工程与重建外科杂志. 2019,15(04)
【文章页数】：7 页
【部分图文】：
流式检测表达效率和荧光强度Fig.4Detectionofexpressioneffi-ciencyandfluorescenceintensitybyflowcytometry
化图A:12hoursafterelectroporation;B:1dayafterelectroporation;C:2daysafterelec-troporation;D:3daysafterelectroporation;E:4daysafterelectroporation;F:5daysafterelectroporation;G:6daysafterelec-troporation;H:7daysafterelectropora-tion;I:ChangesofexpressionefficiencyofEGFPmodRNAinhumanfibroblasts;J:ChangesoffluorescenceintensityofEGFPmodRNAinhumanfibroblasts图4流式检测表达效率和荧光强度Fig.4Detectionofexpressioneffi-ciencyandfluorescenceintensitybyflowcytometryA：光镜图；B：nGFP荧光图；C：DAPI染色；D：Merge图A:Underlightmicroscopy;B:nGFPfluorescentstaining;C:DAPIstaining;D:Mergediagram图5电转nGFPmodRNA后荧光图Fig.5FluorescencediagramafterelectroporationofnGFPmodRNAA：mCherry荧光图；B：EGFP荧光图；C：DAPI染色；D：Merge图A:mCherryfluorescentstaining;B:EGFPfluorescentstaining;C:DAPIstaining;D:Mergediagram图6电转EGFP+mCherrymodRNA后荧光图Fig.6FluorescencediagramafterelectroporationofEGFP+mCherrymodRNAA：EGFP荧光图；B：DAPI染色；C：Merge图；D：流式分析A:EGFPfluorescentstaining;B:DAPIstaining;C:Mergediagram;D:Flowanalysis图7Hela细胞电转EGFPmodRNA后荧光图Fig.7FluorescenceofHelacellsafterelectroporationwithEGFPmodRNA·219·
iRA,deBizemontT,etal.Oculargenetherapy:areviewofnonviralstrategies[J].MolVis,2006,12:1334-1347.[2]PahleJ,WaltherW.Vectorsandstrategiesfornonviralcancergenetherapy[J].ExpertOpinBiolTher,2016,16(4):443-461.[3]TurnbullIC,EltoukhyAA,FishKM,etal.MyocardialdeliveryoflipidoidnanoparticlecarryingmodRNAinducesrapidandtransientexpression[J].MolTher,2016,24(1):66-75.[4]WolffJA,MaloneRW,WilliamsP,etal.Directgenetransferintomousemuscleinvivo[J].Science,1990,247(4949):1465-1468.A：EGFP荧光图；B：DAPI染色；C：Merge图；D：流式分析A:EGFPfluorescentstaining;B:DAPIstaining;C:Mergediagram;D:Flowanalysis图8293T细胞电转EGFPmodRNA后荧光图Fig.8Fluorescenceof293TcellsafterelectroporationwithEGFPmodRNAA：EGFP荧光图；B：DAPI染色；C：Merge图；D：流式分析A:EGFPfluorescentstaining;B:DAPIstaining;C:Mergediagram;D:Flowanalysis图93T3细胞电转EGFPmodRNA后荧光图Fig.9Fluorescenceof3T3cellsafterelectroporationwithEGFPmodRNA·220·
本文编号：3547101
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