SIRT Combines Homologous Recombination, SiteSpecific Integration, and Bacterial Recombineering for Targeted Mutagenesis in Drosophila

GuanjunGao¹,NataliaWesolowska¹,andYikangS.Rong²

LaboratoryofBiochemistryandMolecularBIOLOGy,NationalCancerInstitute,NationalInstitutesofHealth,Bethesda,MD20892,USA

¹Theseauthorscontributedequallytothiswork.

²Correspondingauthor(rongy@mail.nih.gov)

INTRODUCTION

Systematicmutationalanalysisisrequiredforthecomprehensivedecipheringofgenefunction.However,repeatedtargetingofasinglelocusislaborintensiveandhasnotbeenaroutineapproachforstudiesusingmulticellularorganisms.Wehavedevelopedthe"site-specificintegrasemediatedrepeatedtargeting"(SIRT)methodtofacilitatetargetedmutagenesisinDrosophilamelanogaster.InSIRT,homologousrecombinationisusedtoplacealandingsiteforthephagephiC31integraseinthevicinityofthetargetlocus.Allsubsequentgeneticmodificationstothesamegeneareintroducedbyintegrase-mediatedpreciseinsertionofplasmidsdirectlyinjectedintoembryos.ForSIRTmutagenesis,onemustgenerateaseriesofplasmidvectorsthatcontainvariousDNAelementsplacedatdifferentpositionsinthetarget-homologousclone.UnliketrADItionalcloningmethods,SIRTisnotlimitedbytheavailABIlityofconvenientrestrictioncutsites.ThisprotocolpresentsthedetailsofSIRTplasmidconstruction,relyingheavilyonthemethodofbacterialrecombineeringandusinganumberofstreamlinedDNAelements.

RELATEDINFORMATION

FormoreinformationaboutSIRTtargeting,seeGaoetal.(2008).TheproceduredescribedherefortransformingSW102cellscloselyfollows"RecombineeringProtocol1"bySWarming(http://recombineering.ncifcrf.gov/Protocol.asp).ProtocolsareavailableforAgaroseGelElectrophoresis(SambrookandRussell2006a),andPreparationofPlasmidDNAbyAlkalineLysiswithSDS:Minipreparation(SambrookandRussell2006b).

MATERIALS

Reagents

Bacterialstrains:

Standardbacterialcloningstrains(e.g.,DH10BorDH5)

SW102recombineeringcells(availablefromNCI,http://recombineering.ncifcrf.gov)

SW102cellscarryaheat-inducIBLelamBDaredsystem.Itiscrucialtogrowthematorbelow32°Catalltimes(exceptduringinduction).Otherrecombineering-readystrainsarealsoavailable.

DNAligaseand10Xligasereactionbuffer

DNAminipreparationkit(commerciallyavailable)(optional;seeSteps12and18)

DNApolymerasewithproofreadingactivitiesand10Xpolymerasechainreaction(PCR)buffer

Drosophilastocks:

70FLP+70I-SceI(transgeniclinewithheat-inducibleFLPandI-SceIgenes;BloomingtonDrosophilaStockCenter,#6934,#6935)

70FLP10(transgeniclinewithaconstitutivelyactiveFLPgene;BloomingtonDrosophilaStockCenter,#6938)

70I-CreI(transgeniclinewithaheat-inducibleI-CreIgene;BloomingtonDrosophilaStockCenter,#6937)

vasa-phiC31(transgeniclineswithvasa-phiC31onXorIV;www.frontiers-in-genetics.org/flyc31)

White-eyed(w)

FseIrestrictionendonucleaseand10Xreactionbuffer

GenomicDNAtemplatefromDrosophila

Glycerolsolution,sterile,ice-cold(10%v/vinH₂O)(~50mLperbatchofcells)

LB(Luria-Bertani)liquidmediumandsolidplatescontainingtheappropriateantibiotic,atthefollowingconcentrations:

Ampicillin(amp,100µg/mL)

Chloramphenicol(cm,50µg/mL)

Tetracycline(tet,12.5µg/mL)

LRClonaseIIenzymemix(Gateway;Invitrogen)

pCR8/GW/TOPOTACloningKit(Invitrogen)

Plasmidtemplatescontainingthefollowingcloningcassettes:Cm-attP,Cm-attB,andCm-ISceI(availablefromauthorsuponrequest)(seeStep13)

PmeIrestrictionendonucleaseand10Xreactionbuffer

PrimersforPCRamplificationofCm-attP,Cm-ISceI,andCm-attBcloningcassettes(seeSteps13,22,and24)

PrimersforPCRamplificationofdonorfragment(see"GeneralRulesforDesigningPlasmidsforSIRT"andStep1)

pTV2gwends-intargetingvector(availablefromauthorsuponrequest)

Restrictionenzymesforplasmidverification(seeSteps12and18)

SOCmedium

Equipment

Capillarytubingformicroinjection(FHC,#30-30-0)(optional;seeSteps25and32)

Centrifugepresetto0ºC,withrotorfor15-mLtubes(Tomy)

Culturetubes,prechilledonice(15-mL,Nalgene)

DNAsequencingequipment

Electroporator(MicroPulser,BioRad)andcuvettes(1-mm)

EquipmentforDrosophilaculture,includingDrosophilavials

EquipmentforSouthernblotanalysis

Flasks,Erlenmeyer(50-mLor125-mL)

Iceslurry(H₂Oinabucketofice)

Incubatorpresetto30ºC

Incubator,shaking,presetto30ºC

Microinjector(EppendorftraNSJectorp-5246)(optional;seeSteps25and32)

Micromanipulatorformicroinjection(Leica)(optional;seeSteps25and32)

MicroPipettepuller(SutterP-97,Flaming/Brownmicropipettepuller)(optional;seeSteps25and32)

Microscope,dissecting,equippedformicroinjection(optional;seeSteps25and32)

Pipettes,sterile,10-mL

Spectrophotometerandprechilledcuvettes

Thermocycler

Towel,paper

Waterbath,shaking,presetto42ºC

Waterbaths,circulating,presetto36ºCand38ºC(Lauda)

METHOD

VectorConstructions

GeneralRulesforDesigningPlasmidsforSIRT

• Thegenomicfragmentusedfortargeting(the"donor"fragment)shouldcontainenoughhomologytosupporthomologousrecombination.Weroutinelyusefragmentsof5-6kb.ForattPplacement,thedonorcontainsanattPsiteandanI-SceIcutsite.ForphiC31-mediatedintegration,itcontainsanattBsite,aswellasvariousmodificationssuchasmutationsandepitopetags.Forpossibledonorconfigurations,seeFigure1A.

  Viewlargerversion(17K):[inthiswindow][inanewwindow]

  Figure1.DNAelementsforvectorconstruction.(A)Configurationsofthedonorfragmentforvariouspurposes(listedtotheleftofthediagrams).Tomakeadeletion,theattBandattPareplacedatoppositesidesofthedesireddeletion.(B)Cmcassettesforrecombineering.Thearrowsindicateprimerpositions.Forrecombineering,attach50bpofhomologyarms5"totheseprimers.(C)AmapofthepTV2gwtargetingvector.w+:eyeMarkergene.FRT:FLPrecombinasetarget.3"Pand5"P:invertedrepeatsofPelement.

• TheattP/attB(att)site,whichis~250bplong,needstobeplacedsuchthatitdoesnotdisruptexpressionofthetargetgene.Thus,werecommendplacingitinanintergenicregionorwithinalargeintron.
• Theplacementforthe18-bpI-SceIcutsiteisunderfewerconstraints,becauseitisusuallypreciselyeliminatedduringtherecombinationprocessinthetargetingstep(RongandGolic2000;Rongetal.2002).However,ifpossible,placethecutsiteina"nonessential"region,suchastheoneusedfortheattsite,androughlyinthecenterofthedonorfragment(seeFig.1A).
• TherelativepositionbetweenattPandtheI-SceIcutsitescanaffecttheefficiencyatwhichattPisrecoveredinthereductionevent(Rongetal.2002).Asaruleofthumb,placeattP0.5-1kbfromthecutsite.
• Insummary,todesignadonorplasmidforSIRT,startbychoosingapositionfortheattsite.Next,placetheI-SceIcutsite0.5-1kboneithersideoftheattsite,andthenpositionthedonorfragmentsothatthecutsiteisnearthecenter.

GeneratingpTV[donor],theMasterPlasmidCloneforSIRTAflowchartdepictingtheuseofrecombineeringinSIRTisshowninFigure2.

Viewlargerversion(9K):[inthiswindow][inanewwindow]

Figure2.TheuseofbacterialrecombineeringforvectorconstructioninSIRT.Foranoverviewofbacterialrecombineering,seeSawitzkeetal.(2007). 1.Amplifythedonorfragment(5-6kb)fromgenomictemplatesinaPCRusingapolymerasewithproofreadingactivities.UseagarosegelelectrophoresistocheckthespecificityandefficiencyofthePCR(seeAgaroseGelElectrophoresis[SambrookandRussell2006a]).SeeTroubleshooting. 2.ClonethedonorfragmentintothepCR8/GW/TOPOvectorbyTOPOTAcloning,accordingtoInvitrogen’sinstructions.SeeTroubleshooting. 3.SequenceseveralclonesandchooseonethatdoesnotcontainmutationsintroducedbyPCR.Naturalpolymorphismscanbedifferentiatedfrommutationsbylookingforsequencevariationsthatarepresentinmultipleindependentclones. 4.SubclonethedonorfragmentintothepTV2gwvectorusingtheLRclonaseenzymemixaccordingtoInvitrogen’sinstructions.Thisclone,pTV[donor],isthemastercloneusedinsubsequentrecombineeringstepsfortheplacementofvariousDNAelements,suchasattsites,anI-SceIcutsite,andmutations. PreparingCompetentSW102 5.Inoculatea5-mLcultureofLB+tetwithSW102recombineeringcells.Incubatethecultureovernightat30°C. 6.Dilutetheovernightculture50-foldbyadding600µLtoaflaskwith30mLofLB+tet.Incubatewithshakingat250rpmat30°CuntiltheculturereachesanOD600of0.5-0.6(~3h). 7.Chilltheculturebyplacingtheflaskiniceslurryandmixingvigorously.Transferthecellstoprechilled15-mLculturetubes.Centrifugethecellsat5000rpmfor5minat0ºC.Fromthisstepon,trytokeepthecellsat0°Catalltimes. 8.Pouroffasmuchsupernatantaspossiblewithoutlettingthecellswarm.ResUSPendeachcellpelletin1mLofsterile,ice-cold,10%glycerolsolutionbyjostlingthetubesonice.Fillthetubesto10mLwithsterile,ice-cold,10%glycerolsolution,andmixbyinvertingseveraltimes.Centrifugethecellsat5000rpmfor5minat0ºC.Theinitialpelletsarerelativelytight,soresuspensionmaytakeafewminutes. 9.RepeatStep8twomoretimes. 10.Carefullypouroffthesupernatant(thepelletsatthisstageareveryloose),andinvertthetubesonapapertowelforafewsecondsbeforereturningthemtoice.Resuspendthecellsintheresidualliquid,andkeepthetubesoniceuntilelectroporationinStep11. TransformingpTV[donor]intoCompetentSW102 11.Mix1-10ngofpTV[donor]fromStep4with25µLofcompetentSW102cellsfromStep10.Transferthemixturetoprechilledcuvettesandelectroporateaccordingtothemanufacturer’sinstructions.Add500µLofSOCmedium,incubatefor1hat30°C,andplatetheentiremixtureonLB+amp.Incubateovernightat30ºC.KeepanyunusedcompetentSW102cellsin25-µLaliquotsat–80°Cforfutureuse. 12.Prepareminiprepculturesofseveralcoloniesin5mLofLB+amp.Incubatewithshakingovernightat30ºC.IsolateDNAfromtheculturesbyalkalinelysis(seePreparationofPlasmidDNAbyAlkalineLysiswithSDS:Minipreparation[SambrookandRussell2006b])orwithcommerciallyavailableminiprepkits.PerformrestrictiondigestsoftheDNAtoconfirmtransformationofthecells. GeneratingpTV[donor-attP-ISceI],theTargetingVectorforattPPlacementBacterialrecombineeringrequires50bpofhomologyateachsideofthepositionatwhichaDNAcassetteistobeinserted.TherightandleftarmsofhomologyareintroducedaslongPCRprimers. 13.AmplifytheCm-attPcloningcassettefromadilutedplasmidtemplate(seeFig.1B).UsethefollowingpairofPCRprimers: 5"-lefthomologyarm(50bp)-GGCCGGCCCTGTGGAACACC-3"(carriesFseI;homologoustoCmr) 5"-righthomologyarm(50bp)-TCGCGCTCGCGCGACTGACG-3"(homologoustoattP) 14.PrepareSW102cellscarryingpTV2[donor]forinduction: i.Inoculatea5-mLcultureofLB+ampwithSW102recombineeringcellscarryingpTV2[donor]fromStep11.Incubatethecultureovernightat30°C. ii.Dilutetheovernightculture50-foldbyadding600µLtoaflaskwith30mLofLB+amp.Incubatewithshakingat250rpmat30°CuntiltheculturereachesanOD600of0.5-0.6(~3h). 15.Transfer15mLoftheculturetoanewflask.Inducethisaliquotbyincubatinginawaterbathwithshakingat250rpmforexactly15minat42ºC.Returntheremainingcultureto30°Castheuninducedcontrol. 16.Immediatelyafterinduction,washthecells: i.Chilltheculturesbyplacingtheflasksinaniceslurryandmixingvigorously.Transferthecellstoprechilled15-mLculturetubes.Centrifugethecellsat5000rpmfor5minat0ºC.Fromthisstepon,trytokeepthecellsat0°Catalltimes.Treattheinducedanduninducedculturesidentically. ii.Pouroffasmuchsupernatantaspossiblewithoutlettingthecellswarm.Resuspendeachcellpelletin1mLofice-coldH2Obyjostlingthetubesonice.Fillthetubesto10mLwithice-coldH2Oandmixbyinvertingseveraltimes.Centrifugethecellsat5000rpmfor5minat0ºC.Theinitialpelletsarerelativelytight,soresuspensionmaytakeafewminutes.H2Oisusedinsteadofglycerol,becausetheinducedcellscannotbesavedforfutureuse. iii.RepeatStep16.iitwomoretimes. iv.Carefullypouroffthesupernatant(thepelletsatthisstageareveryloose),andinvertthetubesonapapertowelforafewsecondsbeforereturningthemtoice.ResuspendthecellsintheresidualliquidandkeepthetubesoniceuntilelectroporationinStep17. 17.Mix100-300ngoftheCm-attPPCRproductfromStep13with25µLofinducedSW102cellsfromStep16.iv.Transferthemixturetoprechilledcuvettesandelectroporateaccordingtothemanufacturer’sinstructions.Add500µLofSOCmedium,incubatefor1hat30°C,andplatetheentiremixtureonLB+amp+cm.Incubateat30ºC.Coloniesmaytake2dtoappear.SeeTroubleshooting. 18.Prepareminiprepculturesofdouble-resistantcoloniesin5mLofLB+amp+cm.Incubatewithshakingovernightat30ºC.IsolateDNAfromtheculturesbyalkalinelysis(seePreparationofPlasmidDNAbyAlkalineLysiswithSDS:Minipreparation[SambrookandRussell2006b])orwithcommerciallyavailableminiprepkits.PerformrestrictiondigestsoftheDNAbasedontheEcoRIandSmaIsitesintroducedwiththecassette.Theuninducedcontrolshouldproduceveryfewcolonies.Normally,fewerthan50coloniesareexpectedfromtheinducedculture,andmostofthemcarryamixtureofboththecorrectplasmidandtheoriginalpTV[donor]plasmid. 19.To"cleanup"theplasmidmixture,retransformtheminiprepDNAfromStep18intostandardbacterialcloningcells(e.g.,DH10BorDH5)andplateonLB+amp+cm.PickseveralcoloniesandrepeattherestrictiondigestsfromStep18.Mostoftheclonesshouldcontainonlyoneplasmid;thecorrectplasmidisdesignatedpTV[donor-Cm-attP].SequenceseveralclonestoensuretheintegrityofattP.SeeTroubleshooting. 20.DigestafewnanogramsofpTV[donor-Cm-attP]withFseI.Self-ligatethedigestedDNA,transformtheligationreactionsintostandardbacterialcloningcells(e.g.,DH10BorDH5),andplatethetransformedcellsonLB+amp.Pickseveralcolonies,prepareminiprepDNA,andsequencetoverifythelossofCmr.TheplasmidisnowdesignatedpTV[donor-attP].SeeTroubleshooting. 21.RepeatSteps5-12tointroducepTV[donor-attP]intoSW102recombineeringcells.Alternatively,usepreviouslyfrozen,competentSW102cellsandrepeatSteps11and12. 22.SimilartoStep13,amplifytheCm-ISceIcassette(seeFig.1B)withthefollowingpairofprimers: 5"-lefthomologyarm(50bp)-CTATATTACCCTGTTATCCCTA-3"(homologoustoI-SceIcutsite) 5"-righthomologyarm(50bp)-GTTTAAACAGCCAGTATACACTCCGCTA-3"(carriesPmeI;homologoustoCmr). 23.RepeatSteps14-20,butusethepTV[donor-attP]inSW102cellsfromStep21asthestartingplasmid,andusethePCRproductsfromStep22forelectroporation.ToexciseCminStep20,usePmeIinsteadofFseI.Aftersequencing,theresultantpTV[donor-attP-ISceI]plasmidcanbeusedforgermlinetransformationinStep25.SeeTroubleshooting. GeneratingpTV[donor-attB]VectorsforSite-SpecificIntegrationThesevectorsaredifferentfrompTV[donor-attP-ISceI]inStep23inseveralways: •attBreplacesattPintheidenticalposition,unlessthevectorisbeingusedtomakeadeletion,inwhichcaseattBisplacedinanotherposition(seeFig.1). •ThereisnoI-SceIcutsite. •pTV[donor-attB]vectorscarrythedesignedgeneticmodificationtothetargetgene. 24.StartwithSW102cellsthatcarrythepTV[donor]vector,fromStep12.RepeatSteps13-21,butuseCm-attBPCRproductinsteadofCm-attPPCRproduct,andusethefollowingprimers: 5"-lefthomologyarm(50bp)-GGCCGGCCCTGTGGAACACC-3"(carriesFseI;homologoustoCmr) 5"-righthomologyarm(50bp)-CGACATGCCCGCCGTGACCG-3"(homologoustoattB)ThesestepsleadtothecreationofthepTV[donor-attB]plasmid,whichcanbefurthermodifiedusingrecombineeringandcustomizedPCRproductsthatcontainthemodificationsattachedtoanexcisableCmcassette.ThefinalseriesofmodifiedpTV[donor-attB]plasmidswillbeusedformicroinjectioninStep32.SeeTroubleshooting. GeneticCrossesforSIRT PlacementofattPbyEnds-InTargeting Foradetailedpresentationongeneticcrossesforgenetargeting,seeMaggertetal.(2008).ForbackgroundonDrosophilaends-ingenetargeting,seeRongandGolic(2000)andRongetal.(2002). 25.UsepTV[donor-attP-ISceI]fromStep23totransformwhite-eyed(w)fliesbymicroinjection.Alternatively,injectioncanbeperformedbyafee-for-servicecompany.Recoverred-eyed(w+)transformantstoestablishdonorlinesforends-intargeting.ForgeneralinformationonmicroinjectingDrosophilaembryos,seeBachmannandKnust(2008). 26.Mass-matefliesfromtwoorthreedonorlinestofliesfromthe70FLP+70I-SceIlineingroupsoffourorfivepairsofflies.Transfertheparentsevery3d,andheat-shocktheProgenybyincubatingthevialsoflarvaeinacirculatingwaterbathfor1hat38°C.Repeatedheatshocksofthesamevialsarenotnecessary. 27.RecoverfemaleprogenywithboththedonorPelementandthe70FLP,70I-SceIgenes.Thesefliesshouldbemostlywhite-eyed,butsomewillshoweyemosaicism.Mass-matethesefemalestomalesfromtheconstitutive70FLP10lineingroupsofthreeorfourpairs.Transferthecrossesonceafter5d.Asaruleofthumb,aimatsettingup1000crossesspreadoverafewweeks. 28.EstablishlinesfromprogenywithsolideyepigmentationfromStep27.Mapthew+markerinrelationshiptothetargetchromosome.PerformSouthernblotandPCRanalysestoconfirmtargetedeventsandthepresenceofattP. 29.Mass-matetargetedlinestoflieswithaSb-marked70I-CreIgeneonchromosome3ingroupsoffourorfivepairs.Transfertheparentsevery3d,andheat-shocktheprogenybyincubatingthevialsoflarvaeinacirculatingwaterbathfor1hat36°C.RecovermosaicmalescarryingSb70I-CreIindividuallytofemalescarryingchromosome3balancers.Recoverasinglefertile,white-eyed,andSb+(without70I-CreI)malefromeverymosaicfather.Establish50suchstocksforfurthercharacterization.Foratargetlocusonachromosomeotherthanthethirdchromosome,additionaldominantmarkersareneeded. 30.PerformSouthernblotandPCRanalysestoidentifylineswithasingle,intacttargetlocuscarryingthetargetedattPsite.ThesearethemasterstocksforfuturephiC31-mediatedintegrationexperiments. Site-SpecificIntegrationbyphiC31IntegraseThephiC31integrasecanbesuppliedasinvitrosynthesizedmRNAorfromanendogenousphiC31transgene.Werecommendthesecondmethodwithavasa-drivenphiC31insertedonchromosomeXor4(Bischofetal.2007).ForbackgroundonphiC31-mediatedsite-specificintegrationinDrosophila,seeGrothetal.(2004),Batemanetal.(2006),andBischofetal.(2007). 31.Generateastockwithvasa-phiC31andthemasterattPlinefromStep30.Expandthisstockformicroinjectionexperiments. 32.InjectpTV[donor-attB]plasmids(0.5µg/µLinH2O)fromStep24intoembryos.Alternatively,injectioncanbeperformedbyafee-for-servicecompany.Crosssurvivorstowflies,andrecoverpigmentedprogenyasintegrants.VerifythegenomicstructureofintegrantsbySouthernblot,PCR,andsequencinganalyses.Inourhands,100%oftheintegrantstestedhadthecorrectstructure(n>30). 33.RepeatSteps29and30.Thisrepetitionreducesthetargetcopy.Theaboveintegrantscarryatargetduplication,onlyoneofwhichhasthedesiredgeneticmodification.SeeTroubleshooting. 34.Confirmfinalreductioneventswiththedesiredmodificationsbymolecularanalysis. TROUBLESHOOTING Problem:LongPCRisnotproductive. [Step1] Solution:Toincreasetheyieldofamplificationproducts,trythefollowing: 1.Testseveralpairsofprimersfromthesameregion. 2.TreatthetemplategenomicDNAwithRNaseAandphenol. Problem:ThereareveryfewcoloniesaftertheTAreaction. [Step2] Solution:TAcloningdependsonthepresenceofa3"A-overhangonthePCRproducts.Thisoverhangisoftenremovedbyproofreadingpolymerases.Toadd3"A-overhangs,add0.2µLofregularTaqpolymerasedirectlytothecompletedPCR.Incubatethereactionfor10minat72ºC,anduseintheTAcloningreaction. Problem:Theelectroporatorarcs. [Step17] Solution:Arcingiscausedbyexcesssaltinthecell-DNAmixture. 1.Toreducethesaltinthecellsuspension,repeatthecell-washinginStep16anadditionaltwotimes. 2.ToreducethesaltintheDNAcomponentofthemixture,dooneorbothofthefollowingsteps: i.ReducetheamountofPCRproductaddedtothecells. ii.Ethanol-precipitatethePCRproduct,andwashtheprecipitatewithwarm70%ethanol. Problem:Cm-resistantclonesarecarryingtheoriginalplasmidtemplateusedforPCRamplificationofthecloningcassetteinStep13. [Step19] Solution:TominimizetheamountofplasmidtemplateintheDNAusedforelectroporation,digestthetemplatewithDpnIaftercassetteamplificationinStep13.Alternatively,performafirstroundofPCRwiththecassetteprimersonly(withouthomologyarms),dilutethePCRproduct100-to1000-fold,andusethatasthetemplateforthefinalPCRamplification(usingprimerswithhomologyarms).ThePCRreactioncanbedirectlyusedforelectroporation(0.5-4µLofDNAper25µLofcells). Problem:Theplasmidsarerearranged. [Step19] Solution:Largeconstructscanbeunstableinstandardbacterialcloningstrains.UsingStbl2cells(Invitrogen)culturedat30°Cmayhelp. Problem:Allclonesremaindoubleresistant(amprandcmr). [Steps20,23,24] Solution:Considerthefollowing: 1.PerformarestrictiondigesttoconfirmthatCmisexcisable;itispossiblethattheFseIorPmeIsitewasdamagedduringPCR. 2.TheFseIenzymemaybeinactive.StoreFseIat–70ºC,becauseitisunstableat–20ºC. Problem:Reductionfrequencyisverylowand/ormostwhite-eyedeventsdonothaveasingletargetlocus. [Step33] Solution:WeexperiencedanincreaseinreductionfrequencyandfidelitywhenweimplementedanadditionalstepofFLP-mediatedexcisionoftheplasmidbackbonebeforeintroducing70I-CreI(formoredetails,seeGaoetal.2008): 1.CrosstheintegrantsfromStep32totheconstitutive70FLP10line. 2.Heat-shocktheprogenyfor1hat37°Cduringthefirst3doftheirdevelopment. 3.Crosspigmentedprogenyto70I-CreIfliesforreduction. DISCUSSION TargetedmutagenesisthroughhomologousrecombinationhasallowedresearcherstomutateaparticularlocusinDrosophilainanydesiredway.SIRTexpandsthisapproachbytargetingtheattPlandingsitetothelocusofinterest.ThroughphiC31-mediatedintegration,severalgeneticvariantscanthenbegeneratedatthesamelocationwithouthavingtoperformmultiplegenetargetingexperiments.Themethodhasbeenusedsuccessfullyinourlaboratorytocreateanarrayofsixmutantsofthenbslocus,includingaprecisegenedeletion(Gaoetal.2008).phiC31canintegrateDNAfragmentsthatarelargerthan100kbintotheDrosophilagenome(Venkenetal.2006).Inprinciple,SIRTwouldallowtargetedmutagenesistosuchalargeregionadjacenttoanexistingattPsite(seeFig.1A). ThegreatestobstaclethatonefaceswhenusingtheSIRTmethodistheconstructionoftheappropriatevectorsthatcontaincomplexarrangementsofseveralDNAelements.Wesimplifytheprocessbytakingadvantageoftheversatilebacterialrecombineeringmethodology.Thismethodincreasesflexibilityandbypassestheshortcomingsoftraditionalcloning,becauseitdoesnotrelyontheavailabilityofrestrictioncutsitesatthesiteofmodification. ACKNOWLEDGMENTS WethankConorMcMahonandJieChenfortechnicalassistanceduringthedevelopmentofSIRT.WethankDr.DonCourtforprovidingallthenecessaryreagentsforconductingrecombineering.WethankthemembersoftheRongLaboratoryandDr.BrucePatersonatNCIforcommentsontheprotocol. REFERENCES BachmannA,KnustE.2008.TheuseofPelementtransposonstogeneratetransgenicflies.InDrosophilamethodsandprotocols(ed.CDahmann),pp.61–77.Humana,Totowa,NJ. 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