Sepharose4B-CLChromatographyBACDNAPurificationforTransgenicProduction

ThismethodwascontributedbyWesDunnickattheUniversityofMichiganMedicalSchoolinAnnArbor,Michigan.TheapproachistopreparetheDNAwithaslittlemanipulationaspossIBLe,sothattheBACinsertwillremainessentiallyintact,withoutasignificantamountofshearedfragments.TheresultingpreparationhasbothRNAandproteincontamination,whichprobablyaffects(toasmallextent)digestionbyrestrictionenzymes.ThepreparationiscleanedupsubstantiallybythefinalSepharose4B-CLchromatographystep.

ThisprotocolisessentiallyanadaptationofaminiprepfromDavidKohrman’slaboratory,withtheaddedchromatographystepmentionedinYang,Model,andHeintz(NatureBiotechnology15:859-865,1997).WehaveusedtheHeintzmethodformutationofBACswithgreatsuccess.

1.WemaintainourBACsasstreaksonbacterialplatesorascompetentcellsfrozenat-80C.IfthebacteriacontainingaparticularBACdie,thatBACisprobablylostforever;gettingenoughintactDNAtogetherfortransformationofnewbacteriawouldbeverydifficult.

2.Growtwo50mlculturesoftheBACin12.5micrograms/mlchloramphenicol(theresistanceMarkercarriedbyourparticularBAC),startingeachfromasinglecolonyandgrowingovernightat37C.

3.Harvesteachculturebycentrifugationat4000rpminaSorvallSA-600rotorat4C.WetreatthesamplesfromthispointwiththeprecautionsnormallyassociatedwithRNApreparation.Weuseautoclavedtubesandreagents,andweargloves,sinceasmallamountofDNasewouldresultinmanyoftheBACmoleculesbeingcutonceormore.Hence,weharvestin30mlsiliconizedandautoclavedCorextubes.

4.Pouroffthebacterialculturemedia,andplacethetubesonicesothattheremainingfluidcandrainawayfromthebacterialpellet.Afterafewminutes,removethelastofthefluidwithaPasteurpipet.SUSPendandcombinethecellpelletsfromeach50mlcultureintoatotalof4mlsof50mMTris(pH8.0),10mMEDTAwith400micrograms/mlRNaseA.Makesureyouhaveasinglecellsuspension.ClumpsofbacteriawillnotlysewellandwillnotresultinahighyieldofBACDNA.WeuseaPasteurpipettoboth"rub"thecellpelletonthesideoftheCorextubeandtosuspendthecellsbyrepeatedaspiration.

5.Immediatelyaddtoeachcombinedcellpelletfroma50mlculture4mlsof0.2Msodiumhydroxide,1%SDS.Wemakethisfreshfromapelletofsodiumhydroxide,5%SDS,andanappropriateamountofsterilewater(usuallyabout16mlforonesodiumhydroxidepellet).Tolysethebacteria,coverthetubewithParafilmandrotateslowly.Thesolutionshouldbecometranslucent,withoutclumpsofbacteria.Holdatroomtemperaturefor5minutes.

6.Adddropwisetoeachcombinedcelllysate(froma50mlculture)4mlsof3Mpotassiumacetate,pH5.5.Asyouaddthepotassiumacetate,shakethetubegentlytomix.Ideally,agenomicDNA:SDS:proteinprecipitatewillform,andtheremainingsolutionwillbecompletelyclear.Placeonice15minutes.

7.Spinat7500rpmintheSA-600rotorfor15minat4C.Removesupernatant,avoidingthesmallpiecesofprecipitatewhichinevitablyfloatinthesupernatant.Addthesupernatantto7mlof100%isopropanolatroomtemperature.

8.Immediatelyspinat7500rpmintheSA-600rotorfor15minat4C.Wehaveobtainedlargerpelletsbyusingmoreisopropanol,holdingthesampleat-20Cbeforecentrifugation,etc.However,almostallofthelargerpelletisRNAandprotein,whichinhibitsubsequentrestrictionenzymedigestions.Youmaylose20to50%oftheBACDNAusingthisprecipitationapproach,butwhatyouhavewillbehighquality.

9.Washthepellettwicewith5mls70%ethanol.RecovereachtimebycentrifugationintheSA-600rotorat7500rpmfor5minat4C.

10.Removeasmuchoftheethanolsupernatantaspossible,andallowtodryuntilnoethanolmicrodropsarevisible,butthenucleicacidpelletmaystillbeshiny(30to60min).Covereachpellet(originallyfroma50mlculture)with0.15mlof10mMNaCl.Allowthepellettodissolvebyjustsittingatroomtemperaturefor30-60min.IfyouaddhigherconcentrationsofNaClorspermine/spermidineatthispoint,thehighmolecularweightBACDNAwillnotdissolve.

11.Shakethesolutionverygentlytofinishthedissolvingprocess.Add1.5microlitersofasolutionof3mMspermine/7mMspermidine.SpermineandspermidineallowtheBACDNAtoassumea"ball-like"structurethatismuchmoreresistanttomechanicaldamage,butisabletobecutbyrestrictionenzymes.

12.(Usuallynextday)Digest15microliterswithNotIina40microlitervolumeat37Cfor4hoursorso.(Not1releasestheinsertfromthevectorforourparticularBAC.)WeuseyellowtipswithwideopeningstodispensetheBAC.WefindthatwecanvortexthedigestionsolutiononcetomixalltheingredientswithoutshearingtheBACDNA.FractionatethedigestonaCHEFgel,withappropriatesizemarkersandDNAconcentrationstandards.Weusuallyfindthat15microlitersisabout2microgramsofBACDNA,andthatthedigestiongoestocompletion.ThisshouldbeverifiedbyaSouthernblotoftheCHEFgel(useastandardSouthernblotprotocol,butpretreatthegelfor15minwith0.1MHCltoallowtheBACinserttotransferbetter.)WehybridizewithaprobefortheCATgene,whichshouldhybridizetotheBACvectorat11kb,butnottotheinsert.

13.WetesttheDNAfromeach50mlcultureseparately.Thereissomevariationinpreparations,soonealiquotmighthavemoreBACDNA,ordigesttocompletionmoreefficiently.Intheend,thetwopreparationsareusuallyverysimilar,andwepoolthem.

14.Ifthedigestionappearstobecomplete,scaleituptodigest0.15to0.3mloftheBACDNA.Thedigestionsolutionshouldinclude30microMspermineand70microMspermidine.Afterthedigestiscomplete,applyitdirectlytoaSepharose4B-CLcolumnwhichhasbeenequilibratedin0.1MNaCl,10mMTris(pH7.5),0.25mMEDTA(injectionbuffer).Wepourthecolumnina2ml,siliconizedglasspipet,withtheendbrokenoffalittlesothatithasalargerbore.Tobuildthecolumn,weusesiliconized(andbaked)glasswooltostopthebottomofthepipet,whichisalsosiliconizedandbaked.Weattachthebarrelofatenmldisposablesyringetothetopofthepipetasabufferreservoir.ElutetheBACinsertusingthesamebuffer,collectingabouttwelve0.3mlfractions.(Wedothisbyhand;ittakes1-2hours).Weadd3microlitersofthe3mMspermine/7mMspermidinesolutiontoeachtubebeforefractionation,sothattheBACwillbeintheappropriatesolutionimmediatelyasitexitsthecolumn.StoretheBACDNAat4C.

15.Apply35microlitersfromeachfractiontoaCHEFgel,withappropriatesizemarkersandDNAconcentrationsstandards.Wefindthatthevectorelutesinfractions3or4,andtheBACinsert(230kbinourcase)elutesinfractions6or7.(Fig.1--Thisiscounterintuitive,butitworksthiswayeverytime.)Theabsorbanceoffractions9-11areveryhigh(sixtytimeshigherthanfractions6or7),suggestingthattheRNAeluteslate,asitshouldonagelfiltrationcolumn.

16.Fraction6or7havetheBACinsertatabout30microgramsperml,andhencecanbedilutedabout60-fold(withoutfurthermanipulation)forinjectionintoeggs.Ourlatestpreparationyielded74pupsfromtwodaysofinjection.Wescreened70,andfound12founders.OursubsequentSouthernanalysisoftransgenicDNAindicatesthatmostfoundershaveDNAfromthe5’portionandthe3’portionoftheBACinapproximatelyequalcopynumbers,suggestingthatmostcopiesoftheBACenteredgenomicDNAintact.