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1、PEI纳米颗粒基因转染技术方法发布日期:2008-1-8热门指数:2417ABSTRACTThisprotocoldescribesthepreparationofpolyethylenimine(PEI)/DNAnanoparticlesfortargetedgenedelivery.Thisdeliverystrategyimprovestheefticiencyofgenetransferbyenhancingtheentryofgenevectorsintothedesiredcellsandreducinguptakebynontargetcel
2、ls.WedescribeheremethodsfortheconjugationoftargetingpeptidestoPEIs,formationofDNAcomplexesusingtheconjugatedPEIsornonconjugatedPEIstogetherwithtargetingpeptides,andcelltransfectionusingthesecomplexes.Theconjugationstepinvolvestheuseofthesuccinimidyl-4-(N-maleimidomethyl)cyclohexan
3、e-1-carboxylate(SMCC),aheterobifunctionalcross-linker,toformastablebondbetweenPEIandpeptidescontainingthiolgroups・MATERIALSReagents•Deproteinassaykit•DMEMcellculturemediumwith10%fetalbovineserum(FBS)oDMEM(Dulbecco'sModifiedEagle'sMedium)o100units/mlpenicillino100ng/mlstreptomycino
4、2mMglutamineo10%fetalbovineserum(FBS)•Exponentiallygrowingmammaliancells•Lithiumchloride•Luciferaseassayreagents•Dimethylsulfoxide(DMSO)•OptiMEMserum-freecellculturemedium•PEIpolymers(MW600-1000kDa,Fluka;MW750kDa,25kDa,2kDa,and800Da,Sigma-Aldrich;MW1.2,10,or70kDa,Polysciences)•Pep
5、tides,preparedusingconventionalsolid-phase,chemicalsynthesismethod•Phosphatebufferedsaline(PBS)137mMNaCI2.7mMKCI10mMNa2HPO4o2mMKH2PO4Toprepare1literofPBS(Phosphate-bufferedSaline),dissolve8gofNaCI,0.2gofKCI,1.44gofA/&HPO4,and0.24gofKH2PO4in800mlofdistilledH2O.AdjustthepHto7.4(or7.
6、2ifrequired)withHCI.AddH2Oto1liter.Dispensethesolutionintoaliquotsandsterilizethembyautoclavingfor20minutesat15psi(1.05kg/cm2)onliquidcycleorbyfiltersterilization.Storethebufferatroomtemperature.Ifnecessary,PBSmaybesupplementedwith1mMCaCband0.5mMMgC/?Canbemadeasa10xstock.•PlasmidD
7、NAencodingtheluciferasereportergene•ReporterLysisBuffer,5X(Promega),diluteto1XinPBS•Succinimidyl・4・(N-maleimidomethyl)cyclohexane・1・carboxylate(SMCC)•Water,ultrapuresterilizedEquipmentDialysismembranes(molecularweightsizeexclusionspecificationforpurificationofsidereactionandexcess
8、products)•Freezedryer•Luminometer
