Youwillhaveacouplefrustratingsessionswhenyoufirstattemptthistechnique,buteveryoneseemstomasterinjectionafterafewdays,anditworksveryquicklyandreliablyonceyouhavesomeexperience.1.Materialsa)agarosepads:makealotoftheseatatime;theylastforever.UsingaPasteurPipetteplaceadropof2%agaroseinH20ona24x50mmcoverslip.Dropasecondcoverslipontop,whichwillflattentheagaroseintoathinpad.(trytoavoidairbubbles,butafewwon"thurtanything)Whentheagarosehashardened(>5sec)slideoffthetopcoverslip.Usethistopcoverslipasthebottomcoversliptomakethenextpad;itsthincoatingofagarosewillmakethepadsticktoitinsteadofthefreshtopcoverslip.Placethecoverslipsinaboxandcoverwithaluminumfoiltodry.Canleaveoutonthebenchovernight,orbakeinanovenat65·(thesameoneyouusetode-mitewormboxes)for1hour,orbakeinan80·ovenforabout15min.Oncedried,youcanstorethepadsbystickingthembackintheoriginalcoverslipbox.b)10Xmicroinjectionbuffer.

20%polyethyleneglycol,molecularweight6000-8000200mMpotassiumphosphate,pH7.530mMpotassiumcitrate,pH7.5

Mix10mL1MKphosphatepH7.5,5ml300mMKcitrate,pH7.5,10gPEG,and~25mlH20,stir~10mintodissolvethePEG,andaddmoreH20tofinalvolumeof50ml.NotethatthePEGisverynearitssolubilitylimit,sothesolutionmayremaincloudyuntilthesolutionisvol"dto50mlwithH20.Makingthebuffers:1MKphosphatepH7.5

8.7gK2HPO4+50mlH20=1Msolution6.8gKH2PO4+50mlH20=1Msolutionmix32.4ml1MK2HPO4+7.6ml1MKH2PO4togetpH7.5300mMKcitrate,pH7.56.3gcitricacid+~70mlH20addHCLor10NKOHtopHto7.5H20to100ml

c)recoverybuffer:M9buffer.PeopleusedtouseM9plus4%glucose;theglucoseisunnecessaryandonlycausesthesolutiontobecomecontaminated.d)Microinjectionneedles:useamicroscopeslideboxtostorepulledneedles.Puttwostripsofmodelingclayintheboxtoholdtheneedles(pressthemintotheclay,leavingthetiphangingfreeinspace.Weuse"Glass1BBLw/FIL1.0mm4IN"filaments,item#1B100F-4fromWorldPrecisionInstruments,Inc.,(813)371-1003,FAX(813)377-5428.Keeptheseclean,alwaysimmediatelyrecapthetubeafterremovingafilament.WeuseaKopfneedle/pipettepullerModel750,fromDavidKopfInstruments,Tujunga,CA.Turnthemachineon(switchatbackright).Pushthebuttoninthebacktoresettheprogramsandget"0000"displayed.Flicktheleverto"program",press"b",thenpresstheprogramnumberbeingusedthen"e"forenter.Pressing"e"successivelywilltellyoutheparameterssetbytheprogram.Flicktheleverto"run".OntheSternlabmachinewe"reusingprogram10,whichis:heat1=5AU,heat2=0AU,sol=5A,delay=0sec,sol=0.1sec.Ittakes7to15secondstopulltheneedle,althoughthebestneedlesareusuallypulledin11-12seconds.Itisourpracticetonotalloweachindividualtoadjustthespacingofthefilaments(whichisdoneusingthemiddlesetofknobs).Individualscanadjustthemachinetotheirpreferencebyusingdifferentprograms.ThisallowseveryonetoreproducIBLypullneedlestheylikewithoutafuss.Insertaglassfilamentintotheneedlepullerwithouttouchingyourfingerstothepartthatwillbeheated,ortouchingthefilamenttotheheatingelements.Tightenthefilamentinwiththetopknob,DON"TEVERTOUCHTHEMIDDLEKNOB!!!!Slidethebottomunitupalltheway,thentightenthebottomknobsothattheunitisheldsUSPended.Thegreen"ready"lightshouldnowbeon.Closethecover.Pressthe"start"button.Themachinewilltimehowlongittooktopulltheneedle;wantittobe~10.4sec.Carefullyremovethebottomhalfofthefilamentintheboxwithclay,discardthetophalfofthefilament.Ipullabout8needlesatonce.Lately,we"vebeenputtingthefilamentinhigherinthemachineandtakingthetopneedle.e)Microinjectionoil:weuse"halocarbonoilseriesHC-700",P.O.number030B31793,1lbbottle,fromHalocarbonProductsCorporation,130DittmanCt.,N.Augusta,S.C.29841.9/93:thebottlenowsaysCAS#9002-83-9.TheHlocarbonProductsCorporationaddressisP.O.box661,RiverEdge,NJ07661.Phonenumber:803-278-3500.2.MakingtheDNAsolutionWantcleanDNAbufferedatpH7.4inaK+buffer,withnottoomuchNa+init.Upto25-40%DNApreparedusingQiagencolumnsinTE,madeupin1Xinjectionbuffer(seeabove)isokay.IfnecessarytogetridofNa+,canmaketheDNA0.1MKAcpH7.4,add2vol.EtOH,ppt,washin70%EtOH,andresuspendin1Xinjectionbuffer.(Inoneexperiment,Iinjecteda40%TEmixandgot20F1rollersfrom30injectedanimals.ThenIpptedtheDNAandresuspendedininjectionbuffer,injectedagain,andgot70rollersfrom15injectedanimals.WithotherDNAsI"vealsonotedseveral-foldbetterresultsusingDNAininjectionbufferthanIhavetypicallygottenusingDNAinTE.ItthereforeseemsveryworthwhiletouseDNAininjectionbuffer.)TypicalDNAconcentrations:Whentryingtorescueamutantwithcosmidpools,usepRF4(containsthedominantrol-6mutant)at80µg/ml,andeachcosmidtobecoinjectedat20µg/ml.Somecosmidscontainpoisonsequences;inthiscasenotransmittingF1swillbegenerated.MichaelSternhadthisproblemwithsem-5andsolveditbyreducingthecosmidconcentrationto1µg/ml.Forß-galconstructs,coinjectpRF4andtheplasmidconstructeachat80µg/ml.Someconstructsexhibitdominantphenotypiceffects.Thisproblemhasbeensolvedinsomecasesbyloweringtheconcentrationoftheß-galDNAconstructinjected.

3.Settingupthescope,loADIngtheneedle,mounting,andbreakingtheneedle.Setupthescopeandinector:WeuseaZeissAxiovert10microscope;therelevantobjectivesaretheplanneofluor5Xand40X.MountedonthescopeisaNarishigeModelMO-202micromanipulator,onwhichismountedaneedleholderhookeduptoaNarishigeIM300injector.Anitrogengastankishookedupthemicroinjector.Toinject,turnontheN2gastank.Usetheregulatortoadjustthepressurecomingfromthetanktoabout75-80psi;itshouldalreadybesettothisrangeandshouldnotrequireTurnonthemicroinjector.Afterafewsecondsthedisplaywillshowthepressuretheinjectorisreceivingfromthenitrogentank.Itshouldbeabout75-80psi(ifnot,adjusttheregulatorontheN2tanktogetitinthisrange.Nextadjustthepressuresusedforinjection:pressthe"mode"buttontwiceuntilthedisplayshowsthefourpressuresettings(fill,inject,balance,hold).Wedon"tusethefillorholdsettings-ignorethese.Usingthesilverknobsontheinjector,adjust"inject"to18.9psitostartwith.Adjustthebalancepressuretoabout2.5psi.Duringinjections,youwillswitchbetweenthebalancepressureandtheinjectionpressureusingthefootpedal.Thebalancepressure,usedbetweeninjections,isaconstantlowpressurelevelusedtokeeptheinjectionoilfrombackinguptheinjectionneedlebycapillaryaction.Thehigherinjectionpressureisusedtopumptheinjectionsolutionintotheworm.Theinjectionandbalancepressurescanbeadjustedtosuittheneedsoftheparticularneedleyouareusing.Forexample,aneedlewithaverysmallopeningmayrequireahigherinectionpressuretogetanadequateflowoftheinjectionsolution.Afteradjustingthepressures,pressthe"mode"buttonthreemoretimesuntil"action"appearsinthedisplay.Pressthe"baln"buttontoturnonthebalancepressure.Youcannowtogglebetweenthebalanceandinjectpressuresbypressingthefootpedal.Iftheneedlebecomesclogged,youcantryclearingitbypressingthe"clr"button,whichiscurrentlyprogrammedtogivea1secondpulseofhighpressure(thesamepressurecomingfromtheN2tank,~80psi).Loadingtheneedle:Beforeloadingtheneedle,microfugetheDNAsolutionfor10(somesay30)min,topelletparticulatematterthatmightclogtheneedle.Placea0.5µldropofthesolutionontheback(unpulled)endoftheneedle;theneedlecontainsaninnerglassfilamentthatwillwicktheDNAsolutiontotheotherend.Holdingtheneedleuptothelight,youshouldseeliquidatthetipoftheneedle.Lookattheneedleunderthedissectingscopetoseeifthereareanyairbubblestrappedintheliquid.Thesearebad;forsomereasonblowingthemoutthroughthetipoftencausestheneedletoblock,perhapsdirtadherestoandmaybecausestheformationofthebubbleinthefirstplace.Ifthebubbleistrulytinyandneartheneedletip,proceedtobreaktheneedleandblowthebubbleoutthetip.Ifthebubbleisbigger,mounttheneedleonthescope,tilttheneedlesothetipispointingasnearlystraightdownaspossible,andgoawayfor~10ormoreminutes.Hopefully,thebubblewillriseupoutoftheneedletipintotheliquidresevoirabovethetaperoftheneedlewhereitisharmless.Mounttheneedleonthescope:Thewholetoppartoftheaxioverttiltsbacksothatyoucangetattheneedle,andalsosothatyoucanchangeslidesonthestagewithoutriskingtouchingtheneedle.Removetheoldneedlebyunscrewingtheassemblythatholdstheneedle.Beverycarefulhere;thepressurecancausetheneedletoshootoutlikeanarrowhere,sokeepyourfaceetc.outoftheway.Also,therearetwosmallblackrubberOringsintheassembly;makesurethesedon"tfalloutandgetlost.Removetheoldneedleandthrowitout(youwillleaveyourneedleinwhenyou"redone)andinsertyourneedle(backendfirstsoasnotthebreakthetip,obviously),andtightentheneedlebyscrewingtheassemblytogetherwell.Leaveafewmillimetersofthebackendoftheneedlestickingoutthebackendoftheassembly.Thenscrewtheassemblyontotheholderonthescope(don"tdothisastightlyasyouscrewedtogethertheassemblyitself-thatwaywhenyoutaketheneedleoffnexttimethewholeassemblywillcomeoffasaunitandtheneedlewon"tshootoutlikeanarrow.)Turnthethreeknobsonthefinecontrolofthemicromanipulatortothemiddleoftheirrange(5),andusingthecoursecontrols(knobsonthepartofthemicromanipulatormountedonthescope),makesuretheneedleishighenoughsothatwhenyoulowerthetophalfoftheaxiovert,theneedletipwon"tcrashintothestage.Alsousethecoarsecontrolstomovetheneedletipleft/rightforward/backwarduntilitisjustabovetheobjective(willthenseeitglowinginthelightshiningdownfromthecondenser).Breakingtheneedle:Therearetwomethodstobreakoffthetipoftheneedle.The(older?)methodofetchingtheneedletipwithhydrofluoricacidisfallingintodisuseintheHorvitzlab,andtheacidissomewhatdangerous.Themorecommonmethodistophysicallybreaktheneedle.OveraBunsenburnerdrawoutastandard(notmicroinjection)10µlmicropipettetoabout1/5itsstartingthickness.Placeastretchofthedrawnoutpartona24x50mmcoverslip,andputadroportwoofmicroinjectionoilontop.Mountthisontheaxiovert,andusingthe5Xobjective,focusonthemicropipette(seeasharpblacklineontheedgewhenyouarefocusedonthemiddle).Jinsuggestsputtingthemicropipettesothatitdoesn"tgostraightupanddown,butratherisataslight(30·?)angle,soastogetabevelededgeonthemicroinjectionneedle.Usingthefinecontrols,carefullylowertheinjectionneedletowardsthestageuntilitisinthesamefocalplaneasthemicropipette.Atthislowpower,youcan"tseetheactualtip,soyoumayhavetotrythe40Xobjectivetodothis.Again,usingthefinecontrols,slowlymovetheinjectionneedleleftuntilittouchesthemicropipette,andthenpullitback.Tochecktheneedle,pressthefootpedaltolookforflowoutoftheneedle.ShouldseeprettyrapidflowoutoftheneedleusingpressureP2,butnoneatpressureP1.IfthereisnoflowatP2,theneedleisn"tbroken;tryagain.Youwillhavetoseebyexperiencewhattheoptimalflowrateis.Youwanttobeabletofloodthegonadinabout3secondsofflowatP2.YoucanmakefineadjustmentstotheflowbyadjustingP2withtheknob.Whendonebreakingtheneedle,usethefinecontroltolifttheneedleupoutoftheoilinpreparationforinjecting.4.Mountingwormsonaninjectionpad.Takeoutanagarosepadandbreatheonit(about1longbreath)tomoistenit;ifitistoodrythewormswilldryoutanddie-toowetandthewormswon"tstickwell.Placeadropofmicroinjectionoilonthepad.Laythecoversliponthetopofanupsidedownlidofasmallwormplatewithtwostripsoflabtapeacrossit.Thisholdsthecoverslipataboutthesameheightasthewormsonaplatesothatyoudon"thavetofocusaroundtoomuchwhenswitchingbackandforth.Iliketospreadtheoildroparoundwithawormpicksothattheoilisn"ttoodeep.Usingawormpickwithoilonitasglue(wanttominimizetheamountofbacteriayoutransfer)transferadulthermaphroditestotheoildroponthepad.Ifthereisstilladheringbacteria,pushthewormsaroundintheoilwithapickuntilthebacteriacomeoff.Mostpeopleliketousefirstdayadultsthathavealineofabout10eggsinthem;thesehavelargerobustgonads.JinpicksL4animalsandagesthemonedayat20·beforeinjecting.ForEglanimals,youhavetoinjectthemyoungerbeforetheybecomebloated.Asabeginner,stick1-2animalsonapad.Someexpertsdoupto9animalsatonce;Iprefertodoonly2-3tominimizethetime(andthereforetrauma)thatthewormsspenddryingoutonthepadduringinjection.Thetrickistosticktheanimalsdowninthecorrectorientationsothatthevulvaispointingtotheside,andthetwodistalgonadarms(thesyncytialpartwhichyouwillinject)areupagainstthewalloftheanimalontheoppositesidefromthevulva.Youdon"twantthesyncytialgonadtobeontoporunderneaththeanimal.Whentheanimalisintheoil,thesyncytialgonadisvisibleastwoclearareastowardstheanteriorandposterioroftheanimal.Tosticktheanimalright,waituntilitisfloatingintheoilsothatit"sbodyflexuresgosideways,notupanddown,andpattheanimaldownontheagarosepadwithyourpickuntilitisstucktothepad.Avoidstrokingorpattingtheanimalonthehead,whichcankillit;ideallytheanimalwillbefullyimmobilizedexceptforit"shead,whichwillstillbefreeandwiggling.Theanimalsstickbestwhentheyfirsttouchthepad;ifyoufailtostickthemonthefirsttry,itbecomesincreasinglydifficulttostickthemdown;afteryou"verubbedthemalloverthepadapparentlythestuffthatallowsthemtosticktotheagarosebecomeswornoff.Sophisticatescanstickdownawholesetofanimalsinalineinthesameorientationforassemblylineinjecting.Oncetheanimalsareintheoil,workreasonablyfasttogettheprocedureoverwithbeforetheanimalsdehydrate.5.InjectingRotatethetopofthemicroscopeback,placethecoversliponthestage(don"tuseclipstoholditon,youcanremovetheclipsfromthestage).Carefullylowerthetopofthemicroscope,watchingtheneedletoseethatitdoesn"tcrashonthecoverslip(ifyouraiseditabitoffthestagebefore,thiswon"tbeaproblem).Alternatively,Iliketojustraisetheneedlefairlyhighwiththemicromanipulatorinbetweeninjectiions,andthenslidetheoldcoverslipoutfromunderneathit,andslidethenewonein.Usingthe5Xobjectivefindtheworm,makesureitisinthecorrectorientation(vulvaawayfromtheneedle).Canmoveorrotatetheentirestagetomovetheworm,althoughsomeliketomovethecoverslipitself.Itisbesttohavethewormata45·angletotheneedle;thismaximizesthepathlengthfortheneedleinsidethegonad,helpingtomakesureyougetthetipinthegonadinsteadofgoingallthewaythroughandouttheotherside.Carefullylowertheneedleintothefocalplanewiththefineadjuster(atthispoint,youonlyneedtomovetheneedleupanddownwiththemicromanipulator;youalwaysmovetheworm,nottheneedle,up/downleft/right,bymovingthewholestage).Movetothe40Xobjective.Focusonasyncytialgonadarm;thisisrecognizedasasausageshapedclearareasurroundedbyniceroundnuclei.KimbleandSharrock(Dev.Biol.96:189-196(1983))showanexcellentphotographofadissectedgonadthatshouldgiveyouagoodideaofwhattolookforifyou"renewtowormanatomy.Focusonthecenterofthesausagesothatyouseeanicerowofnucleioneithersideofthesausage.Usingthefineadjuster,movetheneedleup/downuntilitsverytipisinfocus.Gentlymovethewormsothatitispressinggentlyagainsttheneedleatapointwherethesyncytialgonadispressedupagainstthebodywall,andsothattheneedletipwillendupinsidethegonadafteritpenetratesthebodywall.Topenetratethebodywall,useyourrightindexfingertogentlytapthemicromanipulatoronthelittleboxwiththeballjointinit(justabovewherethearmtheneedleisonisattached).Thisvibratestheneedlealittlesothatitpuncturestheworm.Hopefullythetipisinthegonadnow;ifitobviouslyisn"tpulloutandtryagain.PressthepedaltostarttheflowofDNA.Ifyou"reinthegonaditshouldbeobvious;asthegonadisfloodeditbloatslikeyou"refillingasausage,andyoucansometimesseethenucleiinthesyncytiumreactingtotheflow.Youwanttoputasmuchliquidinthegonadaspossible;hopefullyitwillflowallthewayaroundturnofthegonad.Eventuallythegonadgetssohugethatliquidstartstoblowouttheanimalthroughtheholethattheneedlewentin;trytoavoidthisbutit"sokifthishappens-youwanttoloadtheanimalabouttothispoint.Agoodruleofthumbistoinjectuntilyouseeagoodamountofliquidhasmadetheturnandhasflowedintotheproximalgonad,andthentoshutofftheflow.Tostoptheflowpressthepedalagainandmovetheanimalawaytogettheneedleout.Melloetal.(EMBOJ.10:3959-3970,1991)showexcellentphotographsofagonadalflood.Usuallyonegonadarmismucheasiertoseewellthantheother,sosomepeopleonlyinjecttheeasygonadarm.Otherstrytoinjectboth.Ifyoumissthegonad,youwillseeliquidfillingthepseudocoelom.Usually,theanimalisok,andyoucanjusttryagain.Itissurprisinglyhardtokillthewormbyjabbingandinjectingitincorrectly.Somepeople(Jin)presstheP1buttonaftereveryinjectiontocleantheneedleandhelpkeepitfromclogging.Eventuallyneedlestendtoclogandmustbechanged.Afteryoufinishaworm,usethefinecontrolstolifttheneedleoutoftheoilbeforemovingthestagetofindanewworm,orremovingthepad.

6.RecoveryPutthepadunderthedissectingscope(ontheinvertedplatelid)andusingaP200pipettemanplaceadropofrecoverybufferontheoildropabovetheworm.Thenpokeawormpickstraightdownthroughtherecoverybufferandoiltotouchtheagarosepadnexttotheworm.Thiswillformachannel,andtherecoverybufferwillformalayerunderneaththeoilinwhichthewormwillfloat.Wormscanbeleftonthepadinrecoverybufferforhours,butyoumightaswellimmediatelymovethemtoplates.(Somesayitisbettertoleavetheminrecoverybufferfor>5minutes-inthiscaseplacethecoverslipinthelidofalargewormplate,andplacetheplateoverittomakeahumidifiedchamber.)Canputupto3injectedwormsonaplate;Ipreferoneworm/plate.Useaslightlydrawnoutandbrokenoffandflamedsmoothlargediametermicropipette(1.5mmdiameterdrawnouttoabouthalfthat)andmouthpipettedthewormsovertoaplate,andseta20·.7.ResultsThreedaysafterinjection,scoretheF1fortheMarkergenephenotype(e.g.rollersifpRF4(rol-6)isused).rol-6animalsareRolevenasyounglarvae,soitistemptingtoscoreandpicktheF1afteronlytwodays:don"tdothis!Theyounglarvaeareverydelicateandyouareliabletokillthembypickingthem.EachRolF1isconsideredanindependenttransformant(evenifseveralcomefromthesameinjectedP0).Therefore,eachRolF1shouldbeplacedonaseparateplatetotrytogetlines.Typicallypeopleinject30P0s(takesjust2hoursifyou"regood),andexpecttoget3-300RolF1.UsuallysomeoftheinjectedP0sgivezeroor1RolF1,mostoftheP0sgive5-15rollers.AsabeginnerIaveraged1-2F1rollersperP0.NowIaverageabout8perP0,andsomepeopledomuchbetter.OftheRolF1,typicallyabout5-30%willtransmitthearray,allowingalinetobeestablished.Typically,linestransmitthearrayto30-80%oftheirProgeny.ThereisvariationamonglinestransformedwiththesameDNA.Forexample,onlyafractionoflinestransformedwithacosmid/pRF4mightgiverescueofamutationinagenefoundwithinthatcosmid,andthestrengthoftherescuewillvaryamonglinesthatdoshowrescue.IntheHorvitzlab,peoplelookat~6linesbeforetheytentativelybelieveanegativeresult.Somelinestransmitatonlyafewpercentpergeneration.Thefrequencyoftransmissionvariesfromanimaltoanimal.Jinsaystobesuretokeeptheselineswheninjectingß-galorGFPconstructs;thelowtransmissionrateoftheextrachromosomalarrayisusefulwhentryingtoselectforchromosomalintegrationofthearray(leadingto100%transmission).Somepeople(me,Mark,Gillian,Jin,Tory)havenotedahighincidenceofmalesintheF1ofinjectedanimals,orinsomeRollines.SincethiswasobservedusingavarietyofdifferentDNAsitislikelyanonspecificeffectofextrachromosomalDNAonchromosomedisjunction,anddoesn"tmeanyourgeneisinvolvedinsexdetermination.Ifyouarerescuingamutant,andusingpRF4asacoinjectionmarker,youmaynoticeanoddeffect;ahighproportion(uptohalf)ofnon-RolF1progenyofrescuedRolanimalsmaythemselvesalsoberescuedforthemutantphenotype.Thisisnotnecessarilyindicativeofmaternaleffectrescueofyourmutant.Rather,itcanbeduetolackofpenetranceoftherol-6dominantalleleand/ormosaicismfortheextrachromasomalarray.Thiscanbedemonstratedbypickingindividualnon-RolrescuedanimalsandshowingthattheythrowRolprogeny.8.AlternatecoinjectionmarkersSometimesitisnotdesirabletohavethedominantRolphenotypeinyourtransgenicworms.Inthesecases,youcanmicroinjectwormscarryingarecessivemarkermutationwitharescuingplasmidforthatgene,alongwithwhateveryourexperimentalDNAis.Thefollowingpropertiesaredesirableforsuchamarkermutant:1)itshouldbeveryeasytoscore,preferablyatallstagesofdevelopment.2)themutantsshouldhavehealthygonadsthatareeasytoinject.Thisissometimesachievedbyusingatsallele,growingtheanimalsatthepermissivetemperaturebeforeinjection,andthenshiftingtothenonpermissivetemperature.3)itshouldbepossibletogetstrongF1rescueofthemutant.4)therescuedanimalsshouldbetrulywildtype.

I"veheardaboutpeopleusingunc-76,dpy-20,andlin-15ascoinjectionmarkers.I"vebeenusinglin-15.Itsmaindrawbackisthatitcanonlybescoredinadults.lin-15(n765ts)animalsareraisedat15·forinjection.Alin-15rescuingplasmidisincludedat50ng/µlintheinjectionmix.I"musingtheplasmidpL15EK,whichIgotfromXiaoweiLu.Thisisan11kbEag1/Nru1rescuingfragmentofcosmidC29B12clonedintopBSKS+cutwithEag1/Kpn1,(usingaKpn1linkerontheNru1end).Afterinjection,thewormsaremovedto20·or25·.At25·thenon-rescuedanimalsareverysick,andthereisastrongselectionfortransgenicworms.At20·thewormsarehealthier,andthetransgenicwormsarerecognizedasnon-Muv.Youshouldwait4daysafterinjectionat20·toscoretheadultF1.EventhoughtheMuvphenotypeonlydevelopsduringtheL4,itappearsthatat25·,n765animalsreachtheL4stagemoreslowlymaternalrescue.Peopleusuallyputlin-15(n765)/+animalsat22.5·inordertoenhancetheMuvphenotypeofthen765homozygotestheythrow.Intwotrialsusinglin-15asacoinjectionmarker,IgotaboutthesamenumberofF1non-MuvanimalsasIusuallygetF1rollersusingrol-6(i.e.about100F1"sfrom20injectedP0s).Howeveronly4%and8%oftheseF1stransmittedtheirarray,whereastypicallymorethan20%ofmyRolF1stransmit.PialiintheBargmannlabsaysshegetsabout15%transmission;she"susingadifferentlin-15plasmidthanIam.Ifindthatlin-15ismuchpreferabletorol-6asamarkerwhentryingtointegrateanarray.TheMuvphenotypeisincrediblyeasytospot,whereasscreeningplatesfortheabsenceofnon-Rolanimals(whichyoudowhentryingtointegratepRF4)isalotharder.