A directional recombination cloning system

PCR 必读

Available online at http://wendang.chazidian.com

Gene408(2008)180–

186

http://wendang.chazidian.com/locate/gene

Adirectionalrecombinationcloningsystemforrestriction-andligation-freeconstructionofGFP,DsRed,andlacZtransgenicDrosophilareporters

ChristinaI.Swanson,TrishHinrichs,LisaA.Johnson,

YingZhao,ScottBarolo?

DepartmentofCellandDevelopmentalBiology,UniversityofMichiganMedicalSchool,AnnArbor,MI,USA

Received23June2007;receivedinrevisedform2November2007;accepted2November2007

Availableonline21November2007ReceivedbyA.J.vanWijnen

Abstract

ThefruitflyDrosophilaisaleadingmodelsystemforthestudyoftranscriptionalcontrolbycis-regulatoryelements,orenhancers.Herewepresentarapid,high-efficiencysystemfordirectionallycloningPCR-amplified,PCR-mutated,orsyntheticenhancersequencesintotheGaneshfamilyofPelementreporterconstructs,whichcontainreportergenesencodingnuclear-localizedeGFP,DsRed,orβ-galactosidase.Thissystem,whichisscalableforeithersmallprojectsorhigh-throughputapproaches,makesuseofbothTOPOandGatewaycloningtechnologiesfordirectional,efficientcloning,withouttheneedforrestrictiondigestionorligationreactions.Itshouldbeespeciallyusefulforthoseresearcherswhowishtotestlargenumbersofputativeenhancers,thosewhoareundertakingdetailedmutationalanalysesofenhancersequences,orthosewhowishtoavoidthedifficultiessometimesencounteredintraditionalcloningstrategies.©2007ElsevierB.V.Allrightsreserved.

Keywords:Pelement;Vector;Gateway;Enhancer;Transcription

1.Introduction

OneadvantageofthefruitflyDrosophilaasamodelsystemforgeneexpressionresearchistheabilitytorapidlyandinexpensivelygeneratelargenumbersoftransgeniclines.Thisallowsforcomprehensiveinvivofunctionalanalysesofenhancers,orcis-regulatoryelements,viatransgenicreporterconstructs.AlthoughtransgenesistechnologyinDrosophila

Abbreviations:GFP,greenfluorescentprotein;DsRed,Discosomaredfluorescentprotein;ChIP,chromatinimmunoprecipitation;PCR,polymerasechainreaction;eGFP,enhancedgreenfluorescentprotein;bp,basepairs;kb,kilobasepairs;MCS,multiplecloningsite;NLS,nuclearlocalizationsignal;dpp;decapentaplegic.

?Correspondingauthor.DepartmentofCellandDevelopmentalBiology,UniversityofMichiganMedicalSchool,3047BSRB,109ZinaPitcherPlace,AnnArbor,MI48109-2200,USA.Tel.:+17347647295;fax:+17347631166.

E-mailaddress:sbarolo@umich.edu(S.

Barolo).0378-1119/$-seefrontmatter©2007ElsevierB.V.Allrightsreserved.doi:10.1016/j.gene.2007.11.003

isaquarter-centuryold,severalrecenttechnicaladvances,mostnotablyChIP-on-chipscreens,enhancerpredictionalgorithms,andthesequencingofmultipleflygenomes,haveincreasedthenumbersofDrosophilaresearcherswishingtotestgenomicDNAsequencesforenhanceractivityinvivo(e.g.,Marksteinetal.,2004;Bermanetal.,2004;ReevesandPosakony,2005;Ostrinetal.,2006;Wrattenetal.,2006;Sandmannetal.,2007).However,thebuildingofenhancer–reporterconstructsandthemutationalanalysisofenhancersequencescanbehinderedbycomplicatedorinefficientDNAcloningsteps,orbyalackofcloningexperienceonthepartoftheresearcher.Ourgroupfrequentlytestsnovelgenomicsequencesinvivoforenhanceractivity,andalsogenerateslargenumbersofenhancerconstructsthathavebeenmutatedwithPCR-basedtechniquessuchasoverlapextensionPCR,or“genesewing”(Hortonetal.,1989,1990;GeandRudolph,1997),orsynthesizeddenovobyassemblyPCR(DillonandRosen,1990;ProdromouandPearl,1992;

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C.I.Swansonetal./Gene408(2008)180–186181

Stemmeretal.,1995).Inordertofacilitatethefunctionaltestingoftheseelements,wehavebuilttheGaneshfamilyofPelementcloningvectors,basedonastrategyforhighlyefficientcloningofDNAsintoreportervectors.Thisstrategy,whichcanbeappliedtotheconstructionoflargeorsmallnumbersofreporters,combinesTOPO®cloningandGate-way®recombinationcloningforthedirectionalinsertionofPCR-amplifiedDNAsintoaninjection-readyreportervector,withveryhighefficiency,andwithouttheneedforrestrictiondigestionorDNAligationreactions.TheGaneshtransgen-esisvectorscontainreportergenesencodingnucleareGFP,nuclearfast-maturingDsRed,ornuclearβ-galactosidase.SuchasystemcouldbevaluabletoanygroupstudyingenhancersinDrosophila,butitshouldbeespeciallyusefulforresearcherswho(a)wishtotestmultiplegenomicsequencesforcis-regulatoryactivity;(b)wishtoundertakeafine-scalemutationalanalysisofanenhancer,requiringlargenumbersofreporterconstructs;(c)areinexperiencedwithtraditionalcut-and-pasteDNAcloningtechniques;or(d)wishtoavoidproblematicorinefficientcloningschemes.2.Materialsandmethods2.1.Ganeshvectorconstruction

Note:FullyannotatedsequencedataforGanesh-G1,-R1,and-Z1areavailableatGenBank(accessionnumbersEF420135,EF420135,andEF420137).

A736bpPCRproductconsistingofcodingsequencefromthekanamycinresistancegeneaminoglycoside3′-phosphotrans-ferasewasamplifiedfrompENTR/D-TOPO(Invitrogen),andmutatedtoremoveapotentialTATAbox,usingthefollowingprimers:

KanSpe:5′-GACTAGTGCATGCGAGCTCGGCGCGCCC-TCATCGAGCATCAAATGAAAC-3′

KanSac3′:5′-CCGGCGCTCCGCGGTGGGTATAAATG-GGCTCGCGATAATGTC-3′.

A196bpfragmentcontainingtheminimalDrosophilaHsp70promoterwasamplifiedfromthepH-Pelicanvector(Baroloetal.,2000)withthefollowingprimers:

HspSac5′:5′-TATACCCACCGCGGAGCGCCGGAGTAT-AAATAGAGGCGC-3′

HspMfe:5′-CCAATTGTCTAGAGGCAGGCCTGCTAG-CTCGACTCTAGCGCGTACCCTAG-3′.

ThesetwofragmentswerethenfusedbyoverlapextensionPCR(Hortonetal.,1990;GeandRudolph,1997)andamplifiedwiththeKanSpeandHspMfeprimerstogeneratea964bpchimericproduct,KanHSP.KanHSPwasthenclonedintotheeGFP-NLSvectorpH-Stinger(Baroloetal.,2000)atSphIandNheIsitestogenerateplasmidKHG.

TheeGFP-NLSreportergeneofKHGwasreplacedwithPCRproductscontainingDsRed.T4-NLS(amplifiedfromthepRedH-Stingervector;Baroloetal.,2004)orlacZ-NLS

(amplifiedfromplasmidC4PLZ;WhartonandCrews,1993)betweenNheIandNotIsitestogenerateplasmidsKHRandKHZ.Thefollowingprimerswereusedtogeneratethe859bpDsRed.T4-NLSfragment:

RFPNhe:5′-GGCTAGCAGGCCTGTTTAAACGATC-CACCGGTCGCCACCATGGCCTCCTCCGAGGACGT-CATC-3′

RFPNot:5′-GGCGGCCGCTTTACTTGTACAAGTA-GCGTCTTCGTTCAC-3′.

Thefollowingprimerswereusedtogeneratethe3551bplacZ-NLSfragment:

lacZNhe:5′-GGCTAGCAGGCCTGTTTAAACGATC-CACCGGTCGCCACCATGAAATATTGCAAATT-TTGCTGC-3′

lacZNot:5′-GGCGGCCGCTTTATTATTATTTTTGACAC-CAGACCAACTGG-3′.

Themini-whitegeneofthePelementvectorCaSpeR-1(Pirrotta,1988)wasexcisedwithPstIandNsiIandreplacedwithashortpolylinkercontainingthefollowingsequence:5′-CTGCAGCAATTGGGGGATGCATGCGGGGGGCG-CGCCGGGGACTAGTCTGC.AG-3′,togenerateplasmidCasP.AnEcoRI/NsiIfragmentfromCaSpeR-1,containingthemini-whitegene,wasthenclonedintoCasPatMfeIandNsiIsitestogenerateplasmidCasW,inwhichmini-whiteisintheoppositeorientationtothatinCaSper-1.AnSphI/SpeIrestrictionfragmentfromKHG,KHR,andKHZincludingthespacer,promoter,andreportergenewasligatedintoCasWtogenerateplasmidsCasG,CasR,andCasZ.

A1731bpPCRproductincludingaGatewaydestinationvectorcassette(Invitrogen)wasligatedintoCasG,CasR,andCasZatSphIandAscIsitestogeneratevectorsGanesh-G1,-R1,and-Z1,whichweretransformedandgrowninccdB-tolerantDB3.1cells(Invitrogen).TheGatewaycassettewasamplifiedfromDNAprovidedintheGatewayVectorConversionSystem(Invitrogen)withthefollowingprimers:

GateSph:5′-GGCATGCATCAAACAAGTTTGTA-CAAAAAAGCTG-3′

GateAsc:5′-GGGCGCGCCATCGAACCACTTTGTA-CAAGAAAGC-3′.

A725-bpsegmentoftheKanR-derivedspacerbetweentheenhancercloningsiteandtheHsp70promoterwasexcisedbyrestrictiondigestionofGanesh-G1,-R1,and-Z1withAscIandSacII,andashortlinkersequence(5′-GGCGCGCCGG-CTCGAGGTACCGCGG-3′)wasinsertedtogeneratevectorsGanesh-G2,-R2,and-Z2.

2.2.pBS-ENTR-TOPOvectorconstruction

Note:ThefullyannotatedsequenceofthepBS-ENTR-TOPOsubcloningvectorisavailableatGenBank(accessionnumberEF411199).

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182C.I.Swansonetal./Gene408(2008)180–186

ThepBS-ENTR-TOPOvectorwasmadebyPCR-amplify-ingaportionofthepolylinkerregionofpBluescriptIISK+(Stratagene),usingprimertagsthatincludedadditionalrestrictionsites,andTOPO-cloningthePCRproductintopENTR/D-TOPO(Invitrogen).ThefollowingPCRprimerswereusedforamplificationfrompBluescript:

pBSSK+mcs5:5′-CACCGCATGCCTCGAGGCCGGCAC-TAGTGGATCCCCCGGGC-3′

pBSSK+mcs3:5′-GGGTACCTCTAGAAGATCTGTCGA-CGGTATCGATAAGCTTGATATC-3′.2.3.Transgenesisandflystocks

Pelementtransformationbyembryoinjectionwasper-formedessentiallyasdescribedbyRubinandSpradling(1982).Aslightlymodifiedprotocolisavailableatsitemaker.umich.edu/barolo/protocols.w1118flieswereusedfortransgenesis.2.4.Tissuepreparation,staining,andmicroscopy

TheeyeimaginaldiscsshowninFig.3A–Cweredissectedfromthird-instartransgeniclarvaeandfixedinformaldehyde.Fig.3AandCshownativefluorescenceofeGFP-NLSandDsRed.T4-NLS,respectively;forthediscshowninFig.3B,arabbitanti-GFPprimaryantibody(Invitrogen)andanAlexa488goatanti-rabbitsecondaryantibody(MolecularProbes)wereused.ThewingimaginaldiscshowninFig.3Dwasdissectedfromathird-instartransgeniclarva,fixedinglutaraldehyde,andstainedforβ-galactosidaseactivity,asdescribedbyRomanietal.(1989).TheembryoshowninFig.3EwasfixedandsubjectedtoRNAinsituhybridizationwithadigoxigenin-

labeledlacZprobe,essentiallyasdescribedbyTautzandPfeifle(1989).Aslightlymodifiedprotocolisavailableathttp://sitemaker.umich.edu/barolo/protocols.

ThefluorescentimagesinFig.3AandCandtheDICbrightfieldimagesinFig.3DandEwereobtainedwithanOlympusBX51microscopeandanOlympusDP70digitalcamera.TheconfocalimageinFig.3BwasobtainedwithanOlympusIX71invertedmicroscopeandanOlympusFV500confocalsystem.

3.Resultsanddiscussion

3.1.CloningDNAsintoaGatewaydonorvector

Therestriction-andligation-freecloningstrategydescribedhereisdesignedtostreamlinethegenerationoftransgenicreporterconstructsforDrosophila,sothatDNAsequencescanbemorequicklyandeasilyassayedforcis-regulatoryactivityinvivo.Inmanycases,thesequencetobetestedisaPCRproduct,havingbeeneitheramplifiedfromgenomicDNAormodifiedwithPCR-basedmutagenesistechniques.ThisPCRproductisdirectionallysubclonedintopENTR/D-TOPO®(Invitrogen),aTOPOcloningplasmidwhichalsoservesasa“donor”vectorforGatewayrecombinationcloning.Directionalityisspeci-fiedbyincludinga5′-CACC-3′sequenceatthe5′endoftheupstreamamplifyingprimer(Fig.1;ChengandShuman,2000;http://wendang.chazidian.comforfurtherdetails).Beforesubcloning,wetypicallygel-purifythePCRproductbyagarosegelelectrophoresisofthePCRreactionfollowedbyextractionofDNAfromagelslicewithUltrafree®-DAspinfiltrationtubes(Millipore),althoughthisstepisnotrequired.Wethenuse4μLofPCRproductinthedirectionalTOPOcloningreaction.For

a

Fig.1.Overviewofrestriction-andligation-freecloningofenhancersequencesandtransferintotheGaneshreportervectors.Steps1–3depictthecloningofDNAsintotheGaneshvectorsbysequentialTOPOandGatewayreactions.SeeSections3.1–3.3andSupplementalProtocolsS1andS2fordetaileddescriptionsofeachcloningstep.Analternativecloningstrategy,inwhichDNAscanbeligatedintothepBS-ENTR-TOPOvectorbytraditionalmethods,isdepictedinsteps1′and2′(bottomleft).TheresultantclonescanbeusedinaGatewayreactionforrecombinationintoGaneshvectors(step3).

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Table1

EfficiencyanddirectionalityofcloningintotheTOPOvectorpENTR/D-TOPO®andtheGateway®vectorsGanesh-G1andGanesh-Z1Vector

Cloningreaction

Cloningefficiencya

Proper

orientationb

183

pENTR/TOPO83%(n=53experiments)d91%(n=48)D-TOPO®c

Ganesh-G1eLRrecombination92%(n=19)100%(n=19)

e

LRrecombination88%(n=21)100%(n=21)Ganesh-Z1

CloningefficiencyreferstotheproportionofDNAminiprepsproducingan

insertbandofapproximatelytheexpectedsizeuponrestrictiondigest,outofallpreparationsgeneratingsufficientDNAforanalysis.b

InsertorientationwasdeterminedbysequencingofminiprepDNA.c

ForpENTR/D-TOPO,eachexperimentrepresentsthecloningofadifferentPCRproductintothevectorviatheTOPOreaction.d

Valuesrepresentthemeanefficiencyofnseparateexperiments,ineachofwhichbetween1and30miniprepsweretested.e

FortheGaneshvectors,eachexperimentrepresentstherecombinationofadifferentenhancersequence,ineitherpENTR/D-TOPOorpBS-ENTR-TOPO,andaGaneshvectorviatheLR

reaction.

a

moredetailedprotocol,seeSupplementaryProtocolS1inOnlineSupplementaryMaterial.

Inourhands,anaverageof83%oftheresultantclonescontainaninsertofapproximatelythecorrectsize,and91%ofthosewereinthedesiredorientation(Table1).Thus,onaver-age,fourminiprepcultureswillyieldatleastonepositiveclone,incorrectorientation,in99.6%ofcases.

3.2.GatewayrecombinationcloningintotheGaneshvectorsThecloningsiteofpENTR/D-TOPOplasmidisflankedbytwoattLrecombinationsequences(Fig.1),whicharerecog-nizedbythebacteriophageλrecombinaseandallowsite-spe-cificrecombinationoftheinsertintoa“destination”vectorcontainingattRsequences(Hartleyetal.,2000).WeuseGateway®LRClonase?EnzymeMix(Invitrogen)toswaptheenhancerfromtheTOPOcloneintooneoftheGanesh

destinationvectors,allofwhichcontainattRsequencesflankingtheenhancercloningsite(Figs.1,2).DNAsequencedifferencesbetweentherightandleftrecombinationsitesensuredirectionalrecombination(http://wendang.chazidian.comforfurtherdetails).Efficiencyofthiscloningstepisimprovedbythefactthatthedonorvectoranddestinationvectorcarrydifferentantibioticresistancegenes(Fig.1),aswellasthefactthattheGaneshvectorscontaintheccdBgene,whichencodesaproteintoxictostandardbacterialstrains(Jafféetal.,1985),andwhichisonlyremovedbyasuccessfulrecombinationevent.“Empty”GaneshvectorsmustbegrowninaccdB-tolerantbacterialstrainsuchasDB3.1(Invitrogen),butoncetheplasmidDNAhasbeenprepared,recombinationcloningreactionscanbetransformedintostandardcompetentcells.WehavefoundthatlinearizingthedestinationvectorpriortoLRrecombinationisnotnecessary;therefore,weuseunmodifiedminiprepormidiprepDNAinourcloningreactions.Foramoredetailedprotocol,seeSupplementaryProtocolS1inOnlineSupple-mentaryMaterial.

Inourhands,theLRreactiontakesplacewith90%efficiency,andproperdirectionalityisobservedin100%ofcasesexamined(Table1).Basedontheseaverages,fourminiprepcultureswillyieldatleastonecorrectclonein99.99%ofcases.Wehavesuccessfullyclonedover100differentDNAsintoGaneshvectors.3.3.Cloningofnon-PCR-amplifiedDNAs

ForcasesinwhichtheDNAsequencetobetestedisnotaPCRproduct,wehavebuiltadonorcloningvectorcontainingapolylinker,ormultiplecloningsite(MCS),containingsitesformanycommonlyusedrestrictionenzymes,andflankedbyattLrecombinationsites.Thisvector,calledpBS-ENTR-TOPO,isdesignedfortraditionalrestriction/ligationcloning,followedbydirectionalGatewayrecombinationcloningintoaGaneshvectororanyotherdestinationvector(Fig.1).Wehavesuc-cessfullyusedthissubcloningvectortogeneratereporterconstructscontainingtraditionallysubclonedDNAfragments,

Fig.2.DiagramoftheGaneshPelementGatewayenhancer-reportertransgenesisvectors.VectorscontaintheTATAboxandinitiatorsequencefromtheDrosophilaHsp70promoter,drivingareportergeneencodingeithernucleareGFP,nuclearfast-maturingDsRed,ornuclearβ-galactosidase.Therecombinationcassette(top)isreplacedbythecis-regulatorysequenceofinterestduringtheGatewayrecombinationevent.SeeSection3.4forfurtherdetails.

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184C.I.Swansonetal./Gene408(2008)180–186

andalsofortestingshortsequencescomposedofannealedoligonucleotides.

3.4.StructureandfeaturesoftheGaneshvectors

TheGaneshvectorscontainthebackboneoftheCaSpeR-1vector(Pirrotta,1988),includingthePelementtranspositionsequences,ampicillinresistancegene,andbacterialoriginofreplication,andalsocontainthemini-whitegenefromCaSpeR-1asamarkerfortransgenesis(Fig.2A).Inordertominimizethepotentialinfluenceofwhiteregulatorysequencesonreportergeneexpression,wehaveinvertedthewhitegeneintheGaneshvectors(Fig.2A).DuringtheLRrecombinationevent,theenhancersequenceinthedonorplasmidisexchangedwithaGatewaycassetteintheGaneshvectorcontainingtheccdBgene(Fig.1);theresultingplasmidvectorcantransformstandardcompetentE.colistrainsandcanbeculturedinampicillin-containingmedia.Thefinalinjectionvectorwillcontaintheenhancersequence,flankedby25-bpattBsequences,upstreamofaminimal,TATA-containingpromoterdrivingthereportergene(Fig.2).Ganesh-G1expressesnucleareGFPasareporter,Ganesh-R1expressesanuclear,fast-maturingformofDsRed(Baroloetal.,2004andreferencestherein),andGanesh-Z1expressesnuclearβ-galacto-sidase(Fig.2).TheshortattBrecombinationsequencesresultingfromtherecombinationeventdonotappeartoaffectthetranscriptionalresponsivenessofthereportervectorsinembryo-nictissuesorimaginaldiscs(Fig.3;additionaldatanotshown).Inordertotesttheabilityofenhancerstoactivatetranscrip-tionatamoderatedistance,wehaveplacedaspacersequencebetweentherecombinationsiteandthepromoter,suchthattheDNAsequencetobetestedisinsertedatposition-837relative

tothetranscriptionstartsite.ForresearcherswhowishtotestDNAelementsatamorepromoter-proximalposition,wehavebuilttheGanesh-G2,-R2,and-Z2vectors,inwhichtheenhancerisinsertedatposition-112.

Ganeshvectorscontainingtestenhancersarecapableofdrivinghighlevelsofreportergeneexpressionintheproperpatterns(Fig.3;additionaldatanotshown),anddonotnotice-ablydifferfromnon-Gatewayvectors,suchasthePelican/Stinger/RedStingerreporters(Baroloetal.,2000,2004),withrespecttoreportergeneresponse.

Althoughthemembersofourgroupclonerelativelysmallnumbersofenhancersequencesatonetime(usually10orfewer),thesystemdescribedherecanbescaledupforahigher-throughputschemedesignedtotestmanypotentialregulatoryelementsinarelativelyshorttime.Thismaybeparticularlyusefultothoseusingcomputationalapproachesormicroarrayscreenstopredictnovelenhancersinthegenome,andwhowishtotestlargenumbersofputativeenhancersinvivo(e.g.,Bermanetal.,2004;Marksteinetal.,2004;ReevesandPosakony,2005;Ostrinetal.,2006;Sandmannetal.,2007).Becausethisen-hancercloningschemeeliminates“cutandpaste”techniquesandinvolvesonlyhigh-efficiencyreactions,evenmolecularnovicesinourgrouphaveavoidedcloningproblemswhenmakingreporterconstructsforthefirsttime.3.5.Doesinsertsizeaffectcloningefficiency?

WehaveexaminedthepotentialeffectofthesizeoftheDNAinsertoncloningefficiency,forbothsteps(TOPO-cloningandGatewayrecombinationcloning)involvedinthegenerationofGaneshreporterclones.Nosignificantrelationship

between

Fig.3.ExpressionpatternsdrivenbyenhancersinGaneshvectors,asdetectedintransgenicanimals.A)GFPfluorescenceintheretinaofaneye-antennaimaginaldisc,dissectedfromatransgeniclarvacarryingthedPax2sparklingenhancer(Floresetal.,2000)inGanesh-G1.B)Nuclear-localizedeGFPincellsofatransgeniceyeimaginaldisc,drivenbyamodifiedversionofthedPax2sparklingenhancerinGanesh-G1anddetectedbyantibodystaining.C)ConfocalimageofDsRed.T4-NLSfluorescenceintheretinaofaneye-antennaimaginaldisc,dissectedfromatransgeniclarvacarryingthesparklingenhancerinGanesh-R1.D)X-galstainingrevealsβ-galactosidaseactivityinawingimaginaldiscfromalarvacarryinganenhancerofthedecapentaplegic(dpp)gene(MüllerandBasler,2000)inGanesh-Z1.E)Wholemounttransgenicembryo,shownindorsalview,inwhichlacZreportergeneexpressionisdetectedbyRNAinsituhybridization.Thisembryocarriesthevisceralmesodermenhancerofthedppgene(Sunetal.,1995)inGanesh-Z1,andshowslacZexpressioninparasegment7ofthevisceralmesoderm.InA-D,theposteriorofthediscistothetopofthepanel;inE,anterioristotheleft.