研究生分子生物學工作基礎新基因研究路線課件

LossofFunction

InhibitionofactivityInhibitionofexpressionInhibitionofActivitySpecificinhibitorsDominantnegativesSpecificantibodiesHistoneDeacetylase(HDAC)inhibitorsHistonedeacetylaseinhibitorsandthepromiseofepigenetic(andmore)treatmentsforcancer.NatureReviewsCancer.2019Vol6.38-51DominantNegativeAmutationwhosegeneproductadverselyaffectsthenormal,wild-typegeneproductwithinthesamecell.Thisusuallyoccursiftheproductcanstillinteractwiththesameelementsasthewild-typeproduct,butblocksomeaspectofitsfunction.Examples:

1.Amutationinatranscriptionfactorthatremovestheactivationdomain,butstillcontainstheDNAbindingdomain.Thisproductcanthenblockthewild-typetranscriptionfactorfrombindingtheDNAsiteleadingtoreducedlevelsofgeneactivation.

2.Aproteinthatisfunctionalasadimer.Amutationthatremovesthefunctionaldomain,butretainsthedimerizationdomainwouldcauseadominatenegativephenotype,becausesomefractionofproteindimerswouldbemissingoneofthefunctionaldomains.

DominantnegativemutantofatranscriptionfactorpolIIPPPInhibitionofExpressionRNAinterference(RNAi)Knockout

RNAINTERFERENCE(RNAi)

AndrewZ.FireandCraigC.Mello

"fortheirdiscoveryofRNAinterference-genesilencingbydouble-strandedRNA".–NobelPrizeinPhysiologyandMedicine2019AndrewFire,SiQunXu,MaryK.Montgomery,StevenA.Kostas,SamuelE.Driver,CraigC.Mello.Potentandspeci?cgeneticinterferencebydouble-strandedRNAinCaenorhabditiselegans

Nature391:806–811,2019RNAiinMammaliancells

>30basepairdouble-strandedRNA

ShortinterferingRNAs<30basepairsPKRinactivePKRactive2’,5’-ASinactive2’,5’-ASactiveDegradationofmRNA(Sequence-specificeffects)(Globaleffects)Cleavage

Inhibitionofproteinsynthesis

DegradationofmRNAsRNAiEndogenousSRC-1RNAi

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19439634365SRC-1mRNASmallinterferingRNA(siRNA)AIB1GRIP1LaminALaminCSRC-1siRNALaminsiRNABufferSRC-1SRC-1siRNALaminAsiRNABufferBuffer

b-gallacZSRC-1siRNALaminsiRNABufferSRC-1siRNALaminsiRNABufferSRC-1siRNALaminsiRNABuffer-++

+Shang,Y.,andBrown,M.Science

295:2465-2468,2019MechanismsInvolvedinRNAiRNaseIIIfamilymembersareamongthefewnucleasesthatshowspecificityfordsRNA.AnalysisoftheDrosophilaandC.elegansgenomesreveals3typesofRNaseIIIenzymes.thecanonicalRNaseIII,whichcontainsasingleRNaseIIIsignaturemotifandadsRNA-bindingdomain(dsRBD;forexampleRNC_CAEEL).Drosha,aDrosophilaenzymethatcontainstwoRNaseIIImotifsandadsRBD.HomelesscontainstwoRNaseIIIsignaturesandanamino-terminalhelicasedomain(DrosophilaCG4792andCG6493;C.elegansK12H4.8)TheDiscoveryofDicerDicerFamilyDrosophila:DicerC.elegans:K12H4.8Arabidopsis:CARPELFACTORY,T25K16.4andAC012328_1)Mammals:Helicase-MOISchizosaccharomycespombe:YC9A_SCHPORNAi

anATP-dependent

ProcessBasedupontheknownmechanismsfortheRNaseIIIfamilyofenzymes,Diceristhoughttoworkasadimericenzyme.CleavageintopreciselysizedfragmentsisdeterminedbythefactthatoneoftheactivesitesineachDicerproteinisdefective,shiftingtheperiodicityofcleavagefrom9–11nucleotidesforbacterialRNaseIIIto22nucleotidesforDicerfamilymembers.MethodsofRNAiknockdowninmammaliancells

SchematicofthesiRNAmediatedRNAinterferencepathway.Afterentryintothecytoplasm,siRNAiseitherloadedontoRISCdirectlyorutilizeaDicermediatedprocess.AfterRISCloading,thepassengerstranddeparts,therebycommencingtheRNAinterferenceprocessviatargetmRNAcleavageanddegradation.siRNAloadedRISCsarealsofoundtobeassociatedwithnucleolusregionandmaybeshuttledinandoutofnucleusthroughanyetunidentifiedprocess.siRNAvs.shRNA:Similaritiesanddifferences.AdvancedDrugDeliveryReviews61(2009)746–759

SchematicoftheshRNAmediatedRNAinterferencepathway.AfterdeliveryoftheshRNAexpressionvectorintothecytoplasm,thevectorneedstobetransportedintothenucleusfortranscription.Theprimarytranscripts(pre-shRNA)followasimilarrouteasdiscoveredfortheprimarytranscriptsofmicroRNA.TheprimarytranscriptsareprocessedbytheDrosha/DGCR8complexandformpreshRNAs.Pre-shRNAsaretransportedtothecytoplasmviaexportin5,tobeloadedontotheDicer/TRBP/PACTcomplexwheretheyarefurtherprocessedtomatureshRNA.MatureshRNAintheDicer/TRBP/PACTcomplexareassociatedwithArgonauteproteincontainingRISCandprovideRNAinterferencefunctioneitherthroughmRNAcleavageanddegradation,orthroughtranslationalsuppressionviap-bodies.TheSpecificityofRNAiifsiRNAselicitaspecificresponse,thenallofthesiRNAsdesignedagainstthesametargetwouldbeexpectedtoproducesimilargeneexpressionsignaturesthesiRNAsdesignedagainstdifferenttargetgeneswouldbeexpectedtoshowdifferentgeneexpressionsignaturesImportantParametersinsiRNATransfectionExperiments

HealthofculturedcellsTransfectionmethod Adherentmammaliancellshavebeentraditionallypre-platedintotissueculturewellsandallowedtoattach,recover,andgrowfor24hpriortotransfection.Reversetransfectionisanalternativemethodoftransfectionwherecellsaretransfectedwhilestillinsuspension(i.e.aftertrypsinizationandpriortoplating).Reversetransfectionisbelievedtoincreasecellexposuretotransfectioncomplexesoftenleadingtogreatertransfectionefficiency.Transfectionconditions LengthofcellexposuretotransfectionagentsshouldbeoptimizedtominimizecellulartoxicityandmaximizesiRNAactivitybyvaryingtheamountoftransfectionagentandcellexposuretimetotransfectioncomplexes.QualityandquantityofsiRNAzh.invitrogen/site/cn/zh/home/References/Ambion-Tech-Support/rnai-sirna/tech-notes/optimizing-sirna-transfection-for-rnai.html

SummaryofDifferentsiRNADeliveryMethodsGeneKnockout

(9-12months)CloningandmappingofmousegenomictargetDNAfromamousecDNAsequence(3-5weeks)Designingandcreatingatargetingvector(6-10weeks)ElectroporationandselectionofEScells(6-10weeks)IdentificationofhomologousrecombinantESclones(2weeks)Expansionofrecombinantclones(1weeks)BlastocystinjectionofrecombinantESclones(4-6weeks)Identificationofgermlinetransmission(10-12weeks)ConfirmationofgermlinetransmissionbyPCR(2weeks)KnockoutvsKnock-inPhenotypicalReadoutsAnimalgrowthanddevelopmentSystem/organ/tissuegrowthanddevelopmentCellularlevelgrowthandproliferationmorphologyandotherbehaviorallychangesmetabolicchangesMolecularLevelchangesintargetgeneexpressionchangesinsignaltransductionpathwayseffectsondownstreamgenesFunctionalAnalysisofGenesSequenceandstructureanalysisExpressionprofilingCellularcompartmentalizationGain-of-functionLoss-of-functionProtein-proteininteractionUpstreamcuesDownstreameffectsProtein-ProteinInteractionCo-immunoprecipitationGSTpull-downFluorescentimagingCo-fractionationFRAPFRETImmunoprecipitationCo-Immnunoprecipitation(co-IP)Co-IPOptimizationStrategiesComplexbindingUselysisandwashbufferswithlowionicstrength(i.e.,<120mMNaCl)thatcontainnon-ionicdetergents(NP-40andTritonX-100),whichareless