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1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMVOLUME234p53Protocolsp53ProtocolsEditedbyEditedbySumitraDebSumitraDebSwatiPalitDebSwatiPalitDebAdenovirusExpressingp5311AdenovirusExpressingp53MatthewHolmes,ElizabethRosenberg,andKristofferValerie1.
2、IntroductionMutationinthep53geneisthemostfrequentlyfoundgeneticabnormalityinhumantumors,resultinginincreasedresistancetochemo-andradiotherapy(1,2).Theunderlyingrationaleforp53-mediatedcancergenetherapyistorestoretheabilityofp53mutanttumorstoundergoap
3、optosis.Whenthewild-typep53geneisdeliveredintotumorcellsofvariousoriginsbyadenoviralvector,thecellsbecomemoresensitivetocisplatinandradiationtherapy(3).Inaddition,adenovirusexpressingp53isagreatlaboratorytoolforthedeter-minationoffunction,forconvenie
4、ntlyproducinglargequantitiesofp53pro-tein,andfordeterminingtheeffectofp53oncellulargeneexpressionusingmicroarraytechnology(4).Thetechnologyformakingrecombinantadenoviruswasdevelopedintheearly1980sbyseveralgroups(5,6).Althoughtheprinciplesformakingrec
5、ombinantadenovirusremainthesametoday,sophisticatedandcleverwaysofconvenientlymakingthesevirusesarecontinuouslyevolving.ThemostcommonlyusedvirusDNAbackbonehastheEIA/E1Bgenesdeleted,whichmakesthisvirusreplicationincompetent,unlesstheseproteinfunctionsa
6、reprovidedintrans.StablytransfectedcelllinesthatexpresstheE1AandE1Bproteinscansupportreplicationofvirus(7,8).TheE3region,whichisnotessentialfortheadenoviruslifecycle,hasalsobeenusedasanintegrationsitefortrans-genesinE1A+adenovirus,inwhichcasethevirus
7、isabletoreplicateinavarietyofdifferentcelltypes(9).ThemaximumDNApackagingcapacityofanadenovirusis105%oftheapprox36-kbadenovirusgenome,makingitpossibletoinsertDNAfragmentsof7.5–8kbinthefirstandsecondgenerationFrom:MethodsinMolecularBiology,vol.234:p53
8、ProtocolsEditedby:S.DebandS.P.Deb©HumanaPressInc.,Totowa,NJ12Holmesetal.adenovirusvectors(10),whichisusuallymorethansufficientfortypicalapplications.MorerecentvectordesignshavetakenadvantageofremovingeithertheE2orE4orbothregions(11,12),makingitpossib
