电击缓冲液对哺乳动物细胞电穿孔效率的影响

100071:,:83RPMI1640DMEMDMEMöF12K2PBSHBSHeBsPBScytomix,3VeroSp2ö0NamalwaX2Gal,:Sp2ö0Namalwacytomix;cytomix:cytomix;;,Q2233EffectofelectroporationbuffersongenetransferefficiencyofmammaliancellsQianFeng,XiaoChengzuInstituteofBiotechnology,AcademyofMilitaryMedicalSciences,Beijing100071AbstractObjective:Toimprovetransfectionefficiencyofmammaliancellsandtosuccessfullytransfectnon2adherentcelllines.Methods:TheefficiencyofgenetransferintothreemammaliancelllinesincludingadherentAfricangreenmonkeykidneycells(Vero),semi2adherentmurinehybridomaSp2ö02AG14cellsandsuspensdedhumanBlymphoblastoidNamalwacellsin8differ2entelectroporationbufferssuchascompletecellculturemedia(RPMI1640,DMEM,DMEMöF12);phosphatebufferedsalinesuchasK2PBS,HBS,HeBs,PBS,andcytomixwerecompared.TransfectionefficiencywasdeterminedbyX2Galstaining;cellviabilitywasdeterminedbytrypanbluestaining.Results:Thebesttransfectionefficiencieswereobtainedincytomixinallthreecelllines,ofwhichNamalwaandSp2ö0cellsarenon2adherentcellsanddifficulttotransfectinotherfrequentlyusedelectroporationmedia.Cytomixsubstantiallyincreasestheviabilityofthecellsafterdeliveryoftheelectricpulse.Conclusion:Cytomixisagoodelectroporationbufferforsus2pendedcelllines.Keywordselectroporation;buffers;cells,cultured,,19675,,,1,2PBS,,,3,,,4,PBSK2PBSHBSHeBscytomixcytomix10119996232BullAcadMilMedSci,Jun1999;Vol23No211.1,DH5A,VeroSp2ö0pcDNA3LacZBNamalwa1.2Vero37DMEMöF12(1÷1)(LifeTechnologies)5%,Namalwa37DMEM(LifeTechnologies),5%,Sp2ö037RPMI1640(LifeTechnologies),5%,1.3DNApcDNA3LacZB2,DH5A100mgöLLB,5DNA,DNA(Qiagen)PEG5DNA1.46Bio2RadGenePulserÊ(2mm):DMEMDMEMöF12RPMI1640;:K2PBS(137mmolöLKCl,2.7mmolöLNaCl,8.1mmolöLNa2HPO4õ12H2O,4mmolöLMgCl2,1.5mmolöLKH2PO4);PBS(1L8gNaCl,0.7gKCl,0.2gKH2PO4,1.15gNa2HPO4);HeBs(20mmolöLHEPES,pH7.05,137mmolöLNaCl,5mmolöLKCl,0.7mmolöLNa2HPO4,6mmolöL);HBS(10mmolöLHEPES,pH7.2,150mmolöLNaCl,5mmolöLCaCl2,Bio2Rad);cytomix(120mmolöLKCl,0.15mmolöLCaCl2,10mmolöLK2HPO4,pH7.6,25mmolöLHEPES,pH7.6,2mmolöLEGTA,pH7.6,5mmolöLMgCl2,2mmolöLATP,5mmolöL):1000LF,150500V20V,1.5X-Gal(5--4--3--B-D-),MacGre2gor2X2GalpcDNA3LacZ,1.69610Ll0.4%,5min,,22.1,8,3X2Gal,3113(%)PBSK2PBSHBSHeBsDMEMöF12DMEMRPMI1640cytomixNamalwaO(4)0(5)0(4)0.01(10)0(1)0.08(3)0.06(11)1.42(5)Vero0.01(4)----0.42(2)0(3)14.7(6)Sp2ö00(6)-----0(6)6.58(5)-1;:PBSK2PBSHBSHeBs;DMEMöF12DMEMRPMI1640;cytomix1,cytomixNamalwa,RPMI164011,,0.06%,DMEMöF12K2PBS15,,HeBs10,Namalwa,,,,cy220119996232tomix,,1.42%,cytomixNamalwa,Namalwa,,cytomixVeroSp2ö0,PBSRPMI16403Vero,PBSDMEMRPMI1640,cytomix2,cytomixSp2ö0,PBSRPMI16406,X2Gal,Sp2ö0,cytomix,,6.58%,cytomixSp2ö02.2,RPMI1640PBScytomix3,VeroSp2ö0Na2malwa31000LF,X2Gal,PBS,cy2tomix300Vcytomix,PBS(1),340V,,cytomix,1Namalwa380V,,cytomix3cytomix,PBS,cytomixCa2+Mg2+,EGTAEDTApH,K+,Na+,,,cytomixATP,,4,,;,cytomixPBS,cytomix,,cytomixVeroSp2ö0Namalwa,2,cy2tomix,1FelgnerPL,GadekTR,HolmMetal.Ahighlyeffi2cientlipid2mediatedDNAtransfectionprocedure.ProcNatlAcadSciUSA,1987,84(9):74132MiyajiH,HaradaN,MizukamiTetal.ExpressionofhumanlymphotoxininNamalwaKJM21cellsadaptedtoserum2freemedium.Cytotechnology,1990,4(2):393ChuG,HayakawaH,BergPetal.ElectroporationfortheefficienttransfectionofmammaliancellswithDNA.NucleicAcidRes,1987,15(3):13104vandenHoffMJB,ChristoffelsVM,LabruyereWTetal.Electrotransfectionwithintracellularbuffer.In:NickoloffJAed.Methodsinmolecularbiology.To2towa:HumanaPressInc.,1997.1855SambrookJ,FritschEF,ManiatisT.Molecularclo2ning:alaboratorymanual.2nded.NewYork:ColdSpringHarborLaboratoryPress,1989.246MacGregorG.Optimizingofelectroporationusingre2portergenes.InChangDC,ChassyBM,SaundersJAetaleds.AcademicPressguidetoelectroporationandelectrofusion.SanDiego:AcademicPress,1992.465(1998-11-30)3012,1