研究生分子生物学工作基础新基因研究路线

LossofFunction,InhibitionofactivityInhibitionofexpression,InhibitionofActivity,SpecificinhibitorsDominantnegativesSpecificantibodies,HistoneDeacetylase(HDAC)inhibitors,Histonedeacetylaseinhibitorsandthepromiseofepigenetic(andmore)treatmentsforcancer.NatureReviewsCancer.2006Vol6.38-51,DominantNegative,Amutationwhosegeneproductadverselyaffectsthenormal,wild-typegeneproductwithinthesamecell.Thisusuallyoccursiftheproductcanstillinteractwiththesameelementsasthewild-typeproduct,butblocksomeaspectofitsfunction.Examples:1.Amutationinatranscriptionfactorthatremovestheactivationdomain,butstillcontainstheDNAbindingdomain.Thisproductcanthenblockthewild-typetranscriptionfactorfrombindingtheDNAsiteleadingtoreducedlevelsofgeneactivation.2.Aproteinthatisfunctionalasadimer.Amutationthatremovesthefunctionaldomain,butretainsthedimerizationdomainwouldcauseadominatenegativephenotype,becausesomefractionofproteindimerswouldbemissingoneofthefunctionaldomains.,Dominantnegativemutantofatranscriptionfactor,polII,InhibitionofExpression,RNAinterference(RNAi)Knockout,RNAINTERFERENCE(RNAi),AndrewZ.FireandCraigC.MellofortheirdiscoveryofRNAinterference-genesilencingbydouble-strandedRNA.NobelPrizeinPhysiologyandMedicine2006,AndrewFire,SiQunXu,MaryK.Montgomery,StevenA.Kostas,SamuelE.Driver,CraigC.Mello.Potentandspecicgeneticinterferencebydouble-strandedRNAinCaenorhabditiselegansNature391:806811,1998,RNAiinMammaliancells,30basepairdouble-strandedRNA,ShortinterferingRNAs30basepairs,PKRinactive,PKRactive,2,5-ASinactive,2,5-ASactive,DegradationofmRNA(Sequence-specificeffects),(Globaleffects),Cleavage,InhibitionofproteinsynthesisDegradationofmRNAs,RNAi,EndogenousSRC-1RNAi,5CCUCAGGGCAGAGAACCAUCUTTTTGGAGUCCCGUCUCUUGGUAGA5,19439634365,SRC-1mRNA,SmallinterferingRNA(siRNA),AIB1,GRIP1,LaminALaminC,SRC-1siRNALaminsiRNABuffer,SRC-1,SRC-1siRNALaminAsiRNABufferBuffer,b-gal,lacZ,SRC-1siRNALaminsiRNABuffer,SRC-1siRNALaminsiRNABuffer,SRC-1siRNALaminsiRNABuffer,-+,Shang,Y.,andBrown,M.Science295:2465-2468,2002,MechanismsInvolvedinRNAi,RNaseIIIfamilymembersareamongthefewnucleasesthatshowspecificityfordsRNA.AnalysisoftheDrosophilaandC.elegansgenomesreveals3typesofRNaseIIIenzymes.thecanonicalRNaseIII,whichcontainsasingleRNaseIIIsignaturemotifandadsRNA-bindingdomain(dsRBD;forexampleRNC_CAEEL).Drosha,aDrosophilaenzymethatcontainstwoRNaseIIImotifsandadsRBD.HomelesscontainstwoRNaseIIIsignaturesandanamino-terminalhelicasedomain(DrosophilaCG4792andCG6493;C.elegansK12H4.8),TheDiscoveryofDicer,DicerFamily,Drosophila:DicerC.elegans:K12H4.8Arabidopsis:CARPELFACTORY,T25K16.4andAC012328_1)Mammals:Helicase-MOISchizosaccharomycespombe:YC9A_SCHPO,RNAianATP-dependentProcess,BasedupontheknownmechanismsfortheRNaseIIIfamilyofenzymes,Diceristhoughttoworkasadimericenzyme.CleavageintopreciselysizedfragmentsisdeterminedbythefactthatoneoftheactivesitesineachDicerproteinisdefective,shiftingtheperiodicityofcleavagefrom911nucleotidesforbacterialRNaseIIIto22nucleotidesforDicerfamilymembers.,MethodsofRNAiknockdowninmammaliancells,SchematicofthesiRNAmediatedRNAinterferencepathway.Afterentryintothecytoplasm,siRNAiseitherloadedontoRISCdirectlyorutilizeaDicermediatedprocess.AfterRISCloading,thepassengerstranddeparts,therebycommencingtheRNAinterferenceprocessviatargetmRNAcleavageanddegradation.siRNAloadedRISCsarealsofoundtobeassociatedwithnucleolusregionandmaybeshuttledinandoutofnucleusthroughanyetunidentifiedprocess.,siRNAvs.shRNA:Similaritiesanddifferences.AdvancedDrugDeliveryReviews61(2009)746759,SchematicoftheshRNAmediatedRNAinterferencepathway.AfterdeliveryoftheshRNAexpressionvectorintothecytoplasm,thevectorneedstobetransportedintothenucleusfortranscription.Theprimarytranscripts(pre-shRNA)followasimilarrouteasdiscoveredfortheprimarytranscriptsofmicroRNA.TheprimarytranscriptsareprocessedbytheDrosha/DGCR8complexandformpreshRNAs.Pre-shRNAsaretransportedtothecytoplasmviaexportin5,tobeloadedontotheDicer/TRBP/PACTcomplexwheretheyarefurtherprocessedtomatureshRNA.MatureshRNAintheDicer/TRBP/PACTcomplexareassociatedwithArgonauteproteincontainingRISCandprovideRNAinterferencefunctioneitherthroughmRNAcleavageanddegradation,orthroughtranslationalsuppressionviap-bodies.,TheSpecificityofRNAi,ifsiRNAselicitaspecificresponse,thenallofthesiRNAsdesignedagainstthesametargetwouldbeexpectedtoproducesimilargeneexpressionsignaturesthesiRNAsdesignedagainstdifferenttargetgeneswouldbeexpectedtoshowdifferentgeneexpressionsignatures,ImportantParametersinsiRNATransfectionExperiments,HealthofculturedcellsTransfectionmethodAdherentmammaliancellshavebeentraditionallypre-platedintotissueculturewellsandallowedtoattach,recover,andgrowfor24hpriortotransfection.Reversetransfectionisanalternativemethodoftransfectionwherecellsaretransfectedwhilestillinsuspension(i.e.aftertrypsinizationandpriortoplating).Reversetransfectionisbelievedtoincreasecellexposuretotransfectioncomplexesoftenleadingtogreatertransfectionefficiency.TransfectionconditionsLengthofcellexposuretotransfectionagentsshouldbeoptimizedtominimizecellulartoxicityandmaximizesiRNAactivitybyvaryingtheamountoftransfectionagentandcellexposuretimetotransfectioncomplexes.QualityandquantityofsiRNA,SummaryofDifferentsiRNADeliveryMethods,GeneKnockout(9-12months),CloningandmappingofmousegenomictargetDNAfromamousecDNAsequence(3-5weeks)Designingandcreatingatargetingvector(6-10weeks)ElectroporationandselectionofEScells(6-10weeks)IdentificationofhomologousrecombinantESclones(2weeks)Expansionofrecombinantclones(1weeks)BlastocystinjectionofrecombinantESclones(4-6weeks)Identificationofgermlinetransmission(10-12weeks)ConfirmationofgermlinetransmissionbyPCR(2weeks),KnockoutvsKnock-in,PhenotypicalReadouts,AnimalgrowthanddevelopmentSystem/organ/tissuegrowthanddevelopmentCellularlevelgrowthandproliferationmorphologyandotherbehaviorallychangesmetabolicchangesMolecularLevelchangesintargetgeneexpressionchangesinsignaltransductionpathwayseffectsondownstreamgenes,FunctionalAnalysisofGenes,SequenceandstructureanalysisExpressionprofilingCellularcompartmentalizationGain-of-functionLoss-of-functionProtein-proteininteractionUpstreamcuesDownstreameffects,Protein-ProteinInteraction,Co-immunoprecipitationGSTpull-downFluorescentimagingCo-fractionationFRAPFRET,Immunoprecipitation,Co-Immnunoprecipitation(co-IP),Co-IPOptimizationStrategies,ComplexbindingUselysisandwashbufferswithlowionicstrength(i.e.,120mMNaCl)thatcontainnon-ionic