Abstract
Functionalgenomicsrequiremanipulationandmodificationoflargefragmentsofthegenome.Suchmanipulationhasonlyrecentlybecomemoreefficientduetothediscoveryofdifferenttechniquesbasedonhomologousrecombination.However,certainlimitationsofthesestrategiesstillexistsinceinsertionofhomologyarms(HAs)isoftenbasedonamplificationofDNAsequenceswithPCR.LargequantitiesofPCRproductslongerthan4–5
kbcanbedifficulttoobtainandtheriskofmutationsormismatchesincreaseswiththesizeofthetemplatethatisbeingamplified.ThiscanbeovercomebyaddingHAsbyconventionalcloningtechniques,butwithlargefragmentssuchasentiregenestheprocedurebecomestime-consumingandtedious.Second,homologousrecombinationtechniquesoftenrequireadditionofantibioticselectiongenes,whichmaynotbedesiredinthefinalconstruct.Here,wereportamethodtoovercomethesizeandselection
Markerlimitationsbyatwo-orthree-stepprocedure.Themethodcaninsertanyfragmentintosmallorlargeepisomes,withouttheneedofanantibioticselectiongene.Wehave
humanizedthemouseluteinizinghormonereceptorgene(
Lhcgr)byinsertinga~55
kbfragmentfromaBACclonecontainingthehuman
Lhcgrgeneintoa170
kbBACclonecomprisingtheentiremouseorthologue.Themethodologyisbasedontherationaletointroduceacounter-selectioncassetteflankedbyuniquerestrictionsitesandHAsfortheinsert,intothevectorthatismodified.Uponenzymaticdigestion,
invitroorin
Escherichiacoli,double-strandbreaksaregeneratedleadingtorecombinationbetweenthevectorandtheinsert.Theproceduredescribedhereisthusanadditionalpowerfultoolformanipulatinglargeandcomplexgenomicfragments.
Withcompletionofthehumanandmousegenomeprojects,alargevolumeofdataforgeneswithunknownfunctionwillbeavailable.Inthepresentandforthcomingeraoffunctionalgenomics,sequencedbacterialartificialchromosomes(BACs)(1),P1vectors(2)andP1artificialchromosomes(PACs)(3)willbeveryusefulintheanalysisofgenefunction,sincetheycanbeusedforconstructionofcomplexvectorsandsubsequentcreationoftransgenic,knockoutandknock-inmice.Thedifficultmanipulationofsuchlargeepisomeswithconventionalcloningtechniqueshasuntilnowlimitedtheircompleteusefulness.However,therecentdevelopmentoftechniquesbasedonhomologousrecombinationinEscherichiacoli(4–8)hasimproved,simplifiedandfacilitatedthemanipulationofbothlargeandsmallvectors,thusbecomingaveryimportanttoolinthedesignoftargetingvectorsandthesubsequentfunctionalanalysisofnewlydiscoveredgenes.
ManipulationofplasmidDNAbyhomologousrecombinationwasfirstreportedinyeast(9)andsincethentechniqueshavebeendevelopedtoincludephage-basedvectorsforconvenientandefficientusein
E.coli.Theuseoftheseplasmidstocarryoutgeneticengineeringhasbeencalled
recombinogenicengineeringor
recombineering(6).Recombineeringexploitsthephagederivedproteinpairs,eitherRecE/RecTfromthe
RacphageorRedα/Redβfromtheλphage,toassistinthecloningorsubcloningoffragmentsofDNAintovectorswithouttheneedofrestrictionenzymesitesorligases(7,8).AlimitationoftheoriginalhomologousrecombinationtechniquewasduetothefactthatbacterialRecBCDnucleasedegradeslinearDNAandinitiallytheeventhadtobestudiedinRecBCD-deficientstrains(7).ThiswasovercomebythediscoverythatRedαandRedβwereassistedbyRedγ,whichinhibitsRecBCDnucleaseactivitymakingitpossibletousethetechniquein
E.coliandothercommonlyusedbacterialstrains(10).Inaddition,therecombinationefficiencywasincreased10–100times(11).Thecombinationofthesethreeenzymes(α,βandγ,orE,Tandγ)inonevectorwasnamedRed/ETrecombinationandthebasicprinciplesofthemethodarethatitrequirestwohomologyregionsof>42
bpinalinearfragment,doublestrandbreaks(DSBs)inbothends,andanotherlinearorcircularplasmidinorderforrecombinationtotakeplace.DSBsareessentialsothatRecEorRedαcanbindanddegradeonechainoftheDNA(5′to3′)andatthesametimeloadRecTorRedβtothesinglestrandchainthatisexposed(7).ThesingleDNAstrandloadedwiththeRecTorRecβrecombinasefindsaperfectmatchsequenceandjoinsthetwosequencesbyeitherchaininvasionorannealing.
However,thissystemrequiresinsertionofhomologyarms(HAs),whichareincludedintheoligonucleotidesthatareusedforamplificationofthePCRproductsusedaslinearsubstratesfortherecombinationevent.ThelimitationsofthePCRreactionwithlongprimersmakeitdifficulttogeneratelargequantitiesoffragmentsthatarelongerthan~4–5
kb,andevenintheeventofbeingabletoPCRlongertemplatestherateandriskofmutations/mismatchesincreaseswiththesizeofthetemplate.IflongerfragmentsofDNAareneededforthecloningproceduresthentheHAsmaybeinsertedwithconventionalrestriction/ligationtechniquesintoprimaryconstructsthatareexcisedandusedforrecombination.Moreover,theinsertionofDNAbyRed/ETcloningishighlyefficientonlyifaselectionmarkergeneisincluded,whichmaybeundesirableinthefinalconstruct.Thishasbeenaddressedbytheuseofelegantselection/counter-selectionsystems(12,13)butthelimitationsofthePCR-sizeoftheinserthasnotyetbeensolved.Insertionoflargefragmentswithrecombineeringmaythusbeatimeconsumingmulti-stepprocess.
BACsandlargeplasmids(e.g.subclonedgenomicfragmentsfromBACsintominimalvectors)areimportanttoolsforthecreationoftransgenicorganismssincetheycontainmostoftheregulatoryelementsneededforoptimalexpression.TherearemanylargemammaliangeneswhichareofthesameorderofmagnitudeorlargerthantheaverageinsertoftheBAClibrariesanditisoftendifficulttofindasingleBACspanningtheentiregeneincludingitscontrollingelements.ItmaythusbedesirabletofuseorexchangefragmentsfromdifferentBACsinordertoobtainlargerfunctionalregionsofDNAortocreatehumanizedmousegenes,wherethegeneofinterestinthemouseBACisexchangedforthehumangenomiccounterpart.FusionoftheregulatoryelementsofoneBACwithanotherBACthatcontainsthecodingregionsofageneofinterestthusbecomesapowerfulstrategytostudytissueorcellspecificgeneexpression.ThiscanbeachievedbyusingoneendofaBACthatoverlapsanareaofanotherashomologyregionsorbyaddingtheHAsintotheinsert-containingvectorbyeitherrecombinationorconventionalcloning(14).However,findingoverlappingBACslimitstheusesofthisstrategy.Moreover,themethodcannotintroduceothernon-overlappingfragmentssuchasthesamegenefromdifferentspeciesoradifferentgeneanditseemstobeefficientonlyifaselectionmarker(Sm)ispresentintheinsertingfragment.
Wereporthereamethod,AssistedLargeFragmentInsertionbyRed/ET-recombination(ALFIRE),wherewehaveovercomeboththesizelimitationsandtherestrictionsofthecloning/subcloningproceduresusedintheRed/ETrecombineeringsystem.WedescribethetransferofaverylargeDNAfragment(~55
kb)fromoneBACintoanotherunrelatedBACbythemeansofamodified
RpsL-counter-selection(CS)system(15)combinedwith
invitroor
invivorestrictionenzymedigestion(16,17).
ALFIREthusrepresentsanimprovementoftoolsthatcanbeusedinordertofacilitateconstructionoflargeandcomplexvectorsutilizedinmolecularbiologyandfunctionalgenomics.
TheRed/ETsystemandthecounter-selectioncassette(RpsL-neo)
TheRed/ETrecombinationsystem(pSC101BADγβαA[tet]),andtheRpsL-neocounter-selectioncassette(CS)werepurchasedfromGenebridges,Dresden,Germany.
BACclones
BACscontainingtheentiremouseorhumanluteinizinghormone/chorionicgonadotropinreceptorgenes(
Lhcgr)werepurchasedfromResearchGenetics(clonesRP23-18D7andRP11-73L19,respectively).IndividualBACcloneswerepropagatedin
E.coliinLuria–Bertani(LB)mediacontainingchloramphenicol(Cm,20
μg/ml)/streptomycin(Stp,75
μg/ml)andpurifiedusingtheLargeconstructkit(Qiagen,Valencia,CA,USA).
PCR
PCRreactionsusingoligonucleotidescarryingHAswereperformedusingaproofreadingpolymeraseandbuffersincludedinthekitTripleMasterMix(Eppendorf,Hamburg,Germany).Thestandardreactionconditionswere96°Cfor3
min,followedby30cycleswith96°C,45
s,57°C45
s,72°C,1–3
min,dependingonthesizeoftheproduct,andafinalelongationof5
minat72°C.AnaliquotofeachPCRproductwasanalysedbygelelectrophoresis.TherestofthePCRproductwasethanol-precipitated,resuspendedinTris–HCl(10
mMpH8.5)andusedfortransformationintoelectrocompetentbacteria.PCRwasalsousedtoscreenforpositiverecombinantsandtoverifycorrectintegration.
Primers
Theshortprimers(20–30
nt)werepurchasedfromTAG,Copenhagen,Denmark,whereasthelongerprimers(60–140
nt)werepurchasedfromThermoElectron,Hamburg,Germany.OligonucleotidesequencesusedfordesignoftheI-SceI-CS-
hmLhcgr-BAC:
CS(I-SceI)tohmLhcgr-F(CCAGCATACTGGCCTAGCCACCGGAGCTCACACTCAGGCTGGCGGGCCATGAAGCAGCGGTTCTCGGCGCTGCAGCTGCTGAAGCTGCTGCTGCTGCTagggataacagggtaatGGCCTGGTGATGATGGCGGGATCG);
CS(I-SceI)tohmLhcgr-R(GTGGACTTTTTTGGGGGGAACATATTTAGATACAATTCAGTAATGCAGTTAACACTCTGTGTAGCGAGTCTTGTCTAGGAGAGCTGTACCTTGACAGTattaccctgttatccctaTCAGAAGAACTCGTCAAGAAGGCG).
Recombination
RecombinationeventswereperformedinelectrocompetentDH10BorHS996cellsharbouringthepSC101BADγβαA[hygro]orthepSC101BADγβαA-I-SceI[amp]plasmidsandusingstandardRed/ETprotocols(8).ElectrocompetentcellswerepreparedasdescribedinthegeneralmanualsfromGenebridges.Electroporationwasperformedat1.35
kV,25
μF,200
ΩusinganEppendorfelectroporator(2510).Bacteriawereincubatedin1
mlLBmediaat37°C,1100
rpmfor1–2
hbeforeplatingonagarplatesconditionedwiththeappropriateantibiotic(s).
Mediaandantibiotics
StandardLBmediaandagarplateswereused.Antibioticswereusedatthefollowingconcentrationsforselectionofhighcopyvectorsonagarplates:ampicillin(Ap)50
μg/ml,kanamycin(Km)25
μg/ml;andinliquidmedia:Ap50
μg/ml,Km50
μg/ml.ForBACsandlowcopyvectors,onplatesandinliquidmedia:chloramphenicol(Cm)20
μg/ml,Ap50
μg/mlandKm15
μg/ml.Tetracycline(Tc)concentrationsforselectionofpSC101BADγβαA[tet]was5and3
μg/mlonplatesandinliquidmedia,respectively.Hygromycine(Hyg)wasusedat30
μg/mlonplatesand15
μg/mlinliquidmediaforselectionofthepSC101BADγβαA[hygro]vectorandstreptomycin(Stp)wasusedataconcentrationof75
μg/ml.
Heat-treatedchlor-tetracycline(cTc)wasusedtoinactivatetheTetrepressor.TheinducercTcwassuspendedinLBataconcentrationof400
μg/mlandautoclavedinapressurecookerfor20
min,thenstoredindarkenvironment.InductionofI-SceIwasachievedbyaddingcTctothegrowingcellsatafinalconcentrationof20
μg/ml.
ConstructionofthepSC101BADγβαA[hygro]vector
ThepSC101BADγβαA[hygro]plasmidwasbasedonthepSC101BADγβαA[tet]vector(GeneBridges)andwasconstructedbystandardRed/ETrecombinationprocedures(8)usingtheprimers
Hygro-F(5′AATGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGGCACCGTGTATGGATAGATCCGGAAAGCCTGAA3′)and
Hygro-R(5′TCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATCTATTCCTTTGCCCTCGGACGAGT3′)inordertoamplifythe
hygromycinphosphotransferasegenefromthepIRESHYG3plasmid(BDBiosciences,PaloAlto,CA,USA).PositiverecombinantswereselectedonLB-agarplatessupplementedwithHyg30
μg/ml.
ConstructionofthepSC101BADγβαA-I-SceI[amp]vector
ThepSC101BADγβαA-I-SceI[amp]vectorwasassembledbyPCRamplificationoftheI-SceIgenetogetherwiththeTet-Repressor,Tet-Promoterand
ampicillinresistancegenesequencesfromthePsiI-digestedpST98ASplasmid(
AF170483)(16)[oligosequences
I-SceItoETHyg-F(AATGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGGCACCGTGTGACCAATTCGGGTCGACTTAT)/
I-SceItoETHyg-R(TCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATTGGTCATGAGATTATCAAAAAGGA)].ThePCRproductwhichcontainedHAsforpSC101BADγβαA[hygro]wasco-transformedwiththepSC101BADγβαA[hygro]vectorintoHS996electrocompetent
E.colicellsharbouringthepSC101BADγβαA[tet]vectorandrecombinationwasachievedusingstandardRed/ETprotocols(8)andselectionwithAp50
μg/ml.
Verificationofpositiverecombinants
PCRwasusedtocheckforcorrectinsertionintoplasmidsorBACsbyusingoneprimerannealingtothevectorandtheothertotheinsert.Insomecasesthefullinsertwasamplifiedbyflankingprimers.PCRreactionswereperformedusingBiotoolspolymerase,Madrid,Spain.Thegeneralconditionsincludedaninitialdenaturationat96°Cfor3
min,followedby30cycleswith95°Cfor45
s,57°Cfor45
sand72°Cfor1–3
min,dependingonthelengthoftheamplifiedproduct.ProductswereanalysedbygelelectrophoresisandwhenneededDNAwasrecoveredorminiprepswerepurifiedandusedforsequencing.Sequencingwasperformedinbothdirectionswhentheproductwaslongerthan1
kborwhenbothintegrationpointswereanalysed.Correctrecombinationwasalsoverifiedbyrestrictionenzymedigestionofpositiveclones.Acompletelistoftheoligonucleotidesequencesusedforscreeningofrecombinantscanbeobtainedfromtheauthorsuponrequest.
SingleBACclonesweregrowninLBmediumconditionedwiththeCm/StpandscreenedbyPCRovertheinsertedareas(themLhcgrpromotertothehLhcgrintron1area;andthehLhcgrexon11tothemLhcgr3′area)usingprimersmLhcgrP-F(CCAGCATACTGGCCTAGCCAC)/hLhcgr1-R(AGTACACAGCGCTCCCGTC)andhLhcgrend-F(CGAAACCCAGAATTAATGGCTA)/mLhcgr3-R(CAATTCACCTGAAGTGCTTAAAGA),respectively.CorrectlyintegratedcloneswerefurtherscreenedbyPCRofareascoveringexons5and6oftheinsertedhLhcgrusingprimersExon5-6-F(GCATGAGGGACTTCTAAATTGC)/Exon5-6-R(TGCTCTTTTTAAGCCAGGAAAG),andbylackofPCRamplificationofeitherthehLhcgr-BAC[primershLhcgrend-F/hLhcgr3-R(GTTTTTAGTGTGGCAGTGGTCA)]orthemLhcgr-BAC[primersmLhcgrend-F(TACCCTTACAGTCATCACTCTGGA)/mLhcgr3-R].
Designingthetoolsforassistedlargefragmentinsertion
ThepSC101BADγβαA[hygro]vectorThepSC101BADγβαA[hygro]plasmidwasassembledbyreplacingthetetgeneofpSC101BADγβαA[tet]withthehygrogeneviastandardRed/ETrecombinationprotocols(8).PlasmidDNAwasisolatedfrompositive(hygromycineselected)recombinantsanddigestedwithNcoItofurtherverifycorrectrecombination(Figure1a)andthreecloneswereadditionallysequencedovertheintegrationsites.TherationalewhythepSC101BADγβαA[hygro]vectorwasconstructedwastofacilitatemanipulationoftargetingvectorsthataresubclonedintothecommonlyusedtetracyclineresistantminimalvectorpACYC184(NewEnglandBiolabs,Beverly,MA,USA).ALFIREcanbeusedformanipulationofanyvectorandtargetingvectorsforconstructionofknockoutorknock-inanimalsareoftenassembledbysubcloningDNAfragmentsfromBACcloneswiththehelpofRed/ETrecombination.Inaddition,thepSC101BADγβαA[hygro]plasmidwasusedasthebasicvectorfortheconstructionofthepSC101BADγβαA-I-SceI[amp]vector.
ThepSC101BADγβαA-I-SceI[amp]vectorThepSC101BADγβαA-I-SceI[amp]vector(SupplementaryFigure1)wasconstructedtopromoteinvivoenzymedigestionfollowedbyrecombinationandtherebyfacilitatingmodificationofBACclonesusingALFIRE.Itwasassembledbyintroducingthetetracycline(cTc)-inducibleI-SceIhomingendonucleasefragmentfromthepST98ASplasmid(16)tothepSC101BADγβαA[hygro]plasmidwithstandardRed/ETprotocols(8).TheentirehygrogenewasthusexchangedwiththeI-SceI-Tet-Rep/Tet-Prom-ampunitgeneratingpSC101BADγβαA-I-SceI[amp].Positive(Apselected)cloneswereverifiedbySspIrestrictionenzymedigestion(Figure1b)andthreeclonesweresequencedoverthemodifiedareas.ThevectordataweresubmittedtoEMBL(AM403094).
ThevectorpSC101BADγβαA-I-SceI[amp]encodestheRedγ,Redβ,RedαandRecAproteinsaswellasthehomingendonucleaseI-SceI.Uponactivationwith
L-arabinoseandchloro-tetracycline(cTc),respectively,theseenzymeswillbeexpressedandcellsharbouringthisplasmidwillbecomecapableofperformingdigestionandrecombinationoftheresultingendscontainingthehomologoussequences.PlasmidsorBACscontainingthe18
bprecognitionsiteofthehomingendonucleaseI-SceIcanbelinearized
invivoandthedouble-strandbreaksthusformedpromoterecombinationtoidenticalhomologysequences.EfficientrecombinationisespeciallyimportantwhenlargeDNAfragmentsarebeingrecombinedsuchasinBACfusionexperiments.
TotestthefunctionalityoftheI-SceImeganuclease,10bacterialcoloniesharbouringtheI-SceI-CS-
hmLhcgr-BACandthepSC101BADγβαA-I-SceI[amp]weregrownovernightat30°CinLBmediawithKm/Cm/Ap.Nextday,threeparalleltubeswith1
mlLBmediaweresetupandinoculatedwith30
μlofeachcultureandincubatedat1100
rpmwiththefollowingconditions(1)cTc/Cm/Ap,37°Cfor1
h;(2)cTc/Cm/Ap,30°Cfor5
h;(3)Cm/Ap,30°Cfor6
h.NoneofthetenclonespropagatedinthecTc-inducedtubesincubatedat37°C,showingthatI-SceIwasexpressedandthattheI-SceI-CS-
hmLhcgr-BACwaslinearizedattheI-SceIsites.Allofthetenclonesgrewinthenon-inducedtubesincubatedat30°CindicatingthattheI-SceIendonucleasewasnotexpressedandthattheI-SceI-CS-
hmLhcgr-BACcloneandthepSC101BADγβαA-I-SceI[amp]vectorcouldcontinuetopropagate.AnothercontrolexperimentwasalsosetupwhereacloneharbouringonlytheI-SceI-CS-
hmLhcgr-BACwasincubatedat37°CinLBmedia(cTc/Cm)for6
h,1100
rpm.ThisclonealsopropagatedindicatingthatcTcisnottoxicto
E.coliattheappointedconcentration.
AssistedlargefragmentinsertionwithRed/ET-recombination(ALFIRE)
ALFIREisbasedontherationalethatHAscanbeeasilyinserteddirectlyintotheacceptingvectoratthesametimewhentheintegrationofaninsert/selectioncassetteisperformed.Weusedlongoligonucleotidescontaining~50baseshomologoustotheacceptorplasmidinsertionsite,~55baseshomologoustotheinsert,andinordertogeneratetheDSBsneededforrecombinationauniquerestrictionsite(RS)sequencewasincludedpriortotheprimerbindingsequence.Thecounter-selectioncassette
RpsL-neo(CS)wasusedforprimaryselectioninordertoreducethebackgroundoftheundigestedacceptingvector.Acceptinganddonorvectorswerelinearized
invitroandtransformedintoRed/ETcompetentbacteriaforrecombinationtotakeplace(
Figure2).WiththisstrategyALFIREcanbeusedefficientlyirrespectiveofthevectororinsertsizeandweshowheretheexchangeofaverylargefragment(~55
kb)fromoneBAC(
hLhcgr)toanotherunrelatedBAC(
mLhcgr).
Intheinitialstep,aCScassetteflankedbyrecognitionsitesofI-SceIandHAstotheinsertandtheacceptorvectorwasdesigned(see
Figure2andSupplementaryFigure2).Theprimerswere~140
ntlongandthiswasthelimitsetbythesuppliertoguaranteegoodqualityprimers.TheI-SceIsequencewaschosenbecausethe18
bplongrecognitionsiteisrareeveningenomicDNA.Infact,the
E.colichromosomedoesnotcontainasingleI-SceIsiteandthemeganucleasehasbeenusedtogenerateDSBsintoforeignDNAamplifiedin
E.coli(16,17).First,theCSflankedbytwoI-SceIrestrictionsites(I-SceI-CS)wasinsertedtothemouse
Lhcgr(
mLhcgr)-BACbyRed/ETrecombinationwiththefollowingprocedure:theI-SceI-CScassettewasamplifiedbyPCRwithprimers[CS(I-SceI)tohmLhcgr-F/CS(I-SceI)tohmLhcgr-R]containing(a)theHAsforinsertion(~50
nt)tothe
mLhcgrpromoterand3′regions;(b)HAs(~55
nt)tosubclonethehuman
Lhcgr(
hLhcgr);(c)the18
ntrecognitionsiteofthehomingendonucleaseI-SceI(TAGGGATAACAGGGTAAT);and(d)theprimersites(24
nt)forthe
RpsL-neo(
Figure2andSupplementaryFigure2).Red/ETcompetentbacteriacarryingthe
mLhcgr-BACweretransformedwiththeI-SceI-CSPCRproductandplatedonLBagarplatessupplementedwithKm/Cm.PositivecloneswerescreenedbyPCR(primersmLhcgrP-F/mLhcgr3-R)andfurtherselectedbytheirinabilitytogrow(negativeselection)onLBagarplatessupplementedwithStp/Cm.Inaddition,theresultingPCRproductwasdigested
invitrowithI-SceI(NEB)inordertocheckforfunctionalityoftheinsertedI-SceIrestrictionsites.OneofthreeclonescontainedamismatchintheI-SceIsequenceleadingtoreducedorabolishedcleavagebythismeganuclease.FivepositivecloneswerefurtherselectedforsequencingoftheentireHAarea,whichneededtobefaultlesstoallowefficientRed/ETrecombination.Fouroutoffivecloneswerewithoutmismatchesandonewasselectedforfurtherexperiments.TheI-SceI-CS-
hmLhcgr-BACconstructcanthusbeusedasageneralPCRtemplate(I-SceI-CS)andtheI-SceIrecognitionsiteswouldnotneedtobeaddedeachtimeintheoligonucleotidesequencesinfutureexperiments,makingtheoligosshorterandeliminatingtheneedofcheckingtheI-SceIsiteseverytime.
InvitrogenerationofDSBsLinearizationofthe
hLhcgr-BACwasperformedbydigestionwithNotI(Promega,Mannheim,Germany),andI-SceI-CS-
hmLhcgr-BACwasdigestedwithI-SceI(NEB)followingthemanufacturer""sinstructions.BothBACswerepurifiedbyphenol:chloroform,precipitatedwithethanolandresuspendedinTris–HClbuffer(10
mM,pH8.5).Approximately3
μgofeachlinearBACwasco-transformedtoRed/ET[hygro]electrocompetentbacteriaandselectionofpositivecloneswasinitiallyperformedonStp/CmagarplatesandfurtherinliquidLBmedia(Stp/Cm).Only2/32PCRscreenedcolonies(6.3%)showedcorrectintegrationofthe~55
kb
hLhcgrinsert(
Table1).Thismightbeduetolow-transformationefficiencyoflonglinearfragments.SinceBACsaredifficulttomanipulate,separateandpurifybygelelectrophoresis,andthe
invitrodigestionresultedinpoorrecombinationefficiency,an
invivodigestionsystemwasalsocreated.
| Table1.EfficiencyforinvitroandinvivoBACfusion(insertionoflongsequences)withALFIRE |
ALFIREinE.coliInvivorecombinationbetweenthe
hLhcgr-BACandtheI-SceI-CS-
hmLhcgr-BACwasachievedbyactivationoftheRed/ETrecombinasesandtheI-SceIhomingendonucleaseexpressedbythepSC101BADγβαA-I-SceI[amp]vectorin
E.coli.DSBsontheI-SceI-CS-
hmLhcgr-BACwereinducedasfollows:Bacterialcultures(1.4
ml)inLBconditionedwithCm/Km/Apfor
E.colicontainingboththeI-SceI-CS-
hmLhcgr-BACandthepSC101BADγβαA-I-SceI[amp]plasmid(
L-arabinoseandcTcinducible)weregrownovernightinashaker(Eppendorf)at30°C,1100
rpm.Nextdayelectrocompetentcellsweremadefrom30
mlculturesfollowingthegeneralmanualofGeneBridges.AtacelldensityofOD0.2,
L-arabinosewasaddedtothemedium(finalconcentration0.2%)andthecultureswerefurthergrownfor45
minat37°C,300
rpm,withthedifferencethatatdifferenttimepoints,inactivatedchloro-tetracycline(cTc)wasadded(finalconcentration20
μg/ml)andthecellsweremadeelectrocompetent.Aftertransformationwith3
μgoflinear(NotIdigested)
hLhcgr-BACcellswereplatedinLB-agarplates(diameter25
cm)containingStp/CmandcTc,15
μg/ml,andincubatedovernightat37°C.Thenumberofcoloniesontheplatewaslow(<100)buttherecombinationefficiencywashighevenwithsuchalongfragmentas~55
kb.Maximumrecombinationefficiency(69%)wasachievedafter15
mininductionwithcTc(
Table1).Thirty-twooftheresultingcolonieswerescreenedbyPCRamplificationoftheintegrationpoints(5′and3′)betweenthe
mLhcgrBACandtheinserted
hLhcgrgene.Twenty-twoof22clonesshowedpositiveresultsinbothends(
Figure3a).Randomly,fivecloneswerepickedandgrownfurthertochecktheirintegrity.Allfiveclonescontainedexons5and6(
Figure3b),whichareinthemiddleoftheinsertedregion,andhadnocontaminationofeitherthe
hLhcgr-BACortheunmodified
mLhcgr-BAC(
Figure3c).Minipreps(5
ml)fromthesameclonesweremadeandtheDNAwassubjectedtorestrictionenzymeanalysis.DigestionwithXhoI/PacIshowedtheexpectedpatternequallyinallclones(
Figure3d).
ThelinearBAC(hLhcgr)couldnotrecombinewiththecircularacceptingBACvector(I-SceI-CS-hmLhcgr)whentheI-SceImeganucleasewasnotinduced.Thelackofrecombinationefficiency(Table2)bythislatterexperimentsuggeststhattherecombinationeventisdependentonboththeDSBsgeneratedbytheI-SceIandtheactivityoftheRed/ETrecombinasesinordertobefullyeffective.ForschematicpresentationoftheuseofALFIREinE.coliseeSupplementaryFigure2.
| Table2.Efficientrecombinationoflargefragmentsisdependentondoublestrandcleavage(I-SceI)oftheacceptingvectortounveilhomologyarmstoRed/ETrecombinasesandinducerecombinationbygaprepair |
TheuseofhomologousrecombinationtogenerateandmanipulatelargefragmentsofDNAhasopenedupnewavenuesinthestudyoffunctionalgenomics.Alimitationoftraditionalrecombinogenicengineeringisthedifficultyofinsertinglargefragmentsintolargeepisomesinasinglestep.Itisnecessarytoovercometheselimitationsinordertofacilitatethemanipulationoflargegenomicsequencesneededfortheproductionoftransgenic,knockoutandknock-inorganisms.
WehaveovercomethemajordifficultyofinsertingHAsintolargefragmentsbyincorporatingthehomologoussequencesintheacceptorvectortogetherwithuniquerestrictionsitestherebypromotinggenerationofDSBsandfacilitatingrecombination.Itisimportanttonote,thatoftensuchrestrictionsitesarepalindromesandthereforethechoiceoftheendonucleaseshouldbecarefullyaddressedtomakesurethattheresultingendshavelittlehomologyinordertoreducevectorre-circularization.Itisknownthataslittleas≥6
nthomologyneareachendmightresultin‘endjoining’(8).HomingendonucleaseshavelongrecognitionsitesthatarenotpalindromesandthepresenceofthesesequencesisrareeveningenomicsizeDNA.WechosethehomingmeganucleaseI-SceIrecognitionsequencesasuniquesitestogenerateDSBsintheacceptorBACcarryingtheHAsfortheinsert.However,whentheentire
hLhcgr(~55
kb)wassubclonedintotheregionbetweenthepromoterandthepolyAofaBACcontainingthemousehomologue(
mLhcgr),theefficiencyafter
invitrodigestion(I-SceI)andisolationwasrelativelylow(6.3%).ThepoorerrecombinationefficiencyismostprobablyduetothelimitationsoftransformingthelargeBACs,inparticulariftheyarelinear,intobacteriaandtheriskofshreddingoftheBACduringtheDNApurificationsteps.Toovercometheseproblemsweconstructedanall-in-oneuniversalvector(pSC101BADγβαA-I-SceI[amp])tobeusedin
E.coli.ThissystemexpressestherecombinasesRedγ,RedβandRedαaswellastheI-SceIhomingendonucleaseunderthetightcontrolof
L-arabinoseandchloro-tetracycline,respectively,andmakesitpossibletolinearizetheacceptorvectorintracellularlyfollowedbyrecombinationbetweenhomologoussequences.Inthiswayonlythe
hLhcgrBACwaslinearresultinginimprovedtransformationefficiencyandrecombination(69%positiveclones).Thus,a10-foldhigherefficiencywasachievedby
invivocomparedto
invitroDSBsgenerationbyI-SceI(
Table1)andthebackgroundwasalsoreducedsignificantlywith
invivoenzymedigestion.
Weshowthatbyusingappropriateuniquerestrictionenzymessites,andsubcloning-HAs,largefragmentscanbecopiedin2or3steps(see
Figure2andSupplementaryFigure2forgeneraloverviewoftheALFIREmethod).ByaddingtheHAsforsubcloningintotheoligosusedforamplificationoftheI-SceI-CScassetteweeliminatedthetime-consuminguseoftransferconstructs.Thepresenceoftheseshortexactsequences(~55
nt)isthussufficienttoresultinahighlyefficientandaccuraterecombination.Wealsoshowthatlongoligos,upto~140
nt,cangeneratesatisfactoryHAsforinsertionandforsubcloning.Alternatively,thedoubleHAscouldbegeneratedbyPCRusingfourshorteroligos(>80
nt)insteadoftwointhesamePCRreaction.
Themethodologydescribedishighlyefficientandaneasyprotocoltofollowinordertoinsert,fuse,exchangeandmanipulatevirtuallyanysizeofDNAorplasmidwithoutlongtemplatePCRandundesiredselectionmarkers.Itistherebyanextratoolinadditiontoexistingmethods(8,16,
