Drosophilagermlinetransformation(NicolasGompel,EvaAylaSchröder,October2015)

Thisprotocol is for thegenetic transformationofDrosophilamelanogaster andotherDrosophilaspecies,using transposable elements. The difference to classical protocols (e.g., Spradling& Rubin, 1982) is thatembryos are not dechorionated. It can be adapted to the injection of CRISPR RNA or DNA, and repairmatricesfortransgeneintegration.

Material

InjectionmixThis protocol allows the injection of various types of nucleic acid mixes. These include

transformationvectors(P-elements,Piggybac,Hermes,Phi-C31)andtheirhelperplasmidsorRNA,CRISPRsgRNA,CRISPRplasmidsandCas9mRNA(orevenCas9protein).The transposable elements used for transformation aremodified such that they can no longermobilizeautonomously.Therecombinase(integrase,transposase)mediatingtheirintegrationisprovidedintrans.Itcanbeproducedendogenously in theembryonicgermlinecellsunder thecontrolofa specificpromoter(bestoption;e.g.,nanospromoter),orprovidedalongwiththevectortobetransformed,intheformofahelperplasmidwiththerecombinasegene(reasonableoption),orintheformoftherecombinasemRNAorprotein(stay-away-fromoption).The transformationof fragmentsof various sizes, fromplasmids to fosmids and toBACs is possible. Theoverall principle is identical, the handling of the injection mix changes. Depending on the type ofintegrationsystem,constructsizeandthesourceofintegrase,thethroughputoftransformationvaries.Mixpreparation

1. Mix ingredients in a 0.5 ml eppendorf tube. Typically prepare 10 to 20 µl of mix. In our hands, the preciseconcentrationofeachreagentwasnotveryimportant,andwefoundthatverylow(0,15µg/µl)concentrationsofthevector were sufficient to grant integration at a reasonable rate. What is very helpful, is to keep the overallconcentrationlow(0.5µg/µlorlower),tokeepthemixfluidityhighandfacilitatetheinjection.

AtypicalmixforPelementorɸC31/attPinjectionwouldbe:

10 µl of construct vector (miniprep, ≃300 ng/µl); 5 µl of helper plasmid if needed (miniprep, ≃300 ng/µl); optional: 1-2 µl of filtered food dye (e.g., McCormick green food coloring; 0.2 µm filter)

2.AllmixesaredilutedinddH2O(otherprotocolsusephosphatebuffers)

3.Vortexmix(forsmallplasmids)orgentlymixwithapipette(forbiggerconstructssuchasfosmidsandBACs)

4.Centrifugemixfor10minutesat14000rpm(bench-topcentrifuge)

5. Carefully harvest most of the supernatant (e.g., 17 µl out of 20 µl) with a pipette and transfer it to a cleaneppendorftube.Usethiscentrifugedmixtofillneedles.Themixcanbekeptat4°Cforweeks.

[Alternativeto4-5,runyourmixthroughaMillex-GVcolumn(Millipore)]

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FliesFlystocksshouldbeamplifiedinadvance,fortworeasons:(i)togetenougheggsforinjectionand

(ii)tocollectvirginfemalesforsubsequentcrosseswiththeinjectedflies(G0).

Thespecificstocksvarywithwhatneedstobeinjected,andwhattransformationmarkerisused.

Transfer ̴̴300mature flies toanegg-lay cage(see picture below; e.g., Flystuff Cat #59-100), 1-3days prior to the injection to accustom the flies.Placingthecageunderadesklampcontrolledwithaprogrammabletimerswitchhelpstohaveregulareggcollections. Change the egg-lay cap (e.g., agar-molasses: see picture below, recipe to the right) atleast once per day; spread some yeast paste in thecenter.

The conditions for embryo collection should be such that at least 200 freshly laid embryos areavailableevery20-30minwheninjecting(seepicturebelow).

NeedlesPulling

–Donotoperatetheneedlepullerwithoutadequatetraining!–

Thequalityofneedlesiscriticalforthesmoothnessofthe injection process, the embryo survival and thetransformation efficiency. Needles should be pulled on anySutter instruments brandhorizontalpuller (seepictureontheright; e.g., P-1000 Flaming/Brown Micropipette Puller).Needles are pulled from 1.0 mm OD borosilicate capillarieswithomegadotfiber(e.g.,Sutterinstruments#BF100-50-10).The settings for the design of optimal needles are linked tothecapillarybatchandtheshape/type/positionoftheheatingfilament.Theseparametersdefinearampvalue–themeltingpoint of the capillaries under these conditions. A specificfunctionofthepullercalculatesthisrampvalue.Refertothemanualofyourpullerfordetails.

The ideal needle to inject an embryo in its chorion should have a short taper, a thin tip and nodiscontinuityorstep.Aneedlewithalongtaperwillbendagainsttheembryoandwillnotgothroughthe

Egglaycaps-500mltapwater,boil-25 g agar, dissolve completely whilemixing fromtimetotime-200mlmolassesor200mlgrapejuice-[optional: When cooled below 70°C: 6 mlTegosept/Nipagin solution (10 g in 100 ml 95%ethanol)]-Pourplates(ca.50)-Storesolidifiedplateswithlidat4°C.

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chorion. A needle with a blunt tip will cause damages and cell leakage, thereby increasing embryoniclethality.Differentparametersinfluencetheshapeandpropertiesoftheneedleandtheeffectproducedbychanging anyof them (heat, velocity of pull, pressureof gas flow, numberof steps) is not intuitive. Themost useful documentation to design proper needles is the pipette cookbook provided by SutterInstruments. In addition, Miller et al. (2002) published a very useful guideline for designing suitableneedlesspecificallyforDrosophilaeggsintheirchorion.

For instance,asuccessfulneedletypethatwehavebeenusingfora longtime,wasobtainedusingaboxfilament (Sutter InstrumentFB230B)with thickwalledglasscapillaries (Sutter instruments#BF100-50-10,ramp=625)andthefollowingparameters:

Pressure: 300 Heat: 505 Pull: 20 Velocity: 60 Delay: n/a Time: 250 Looping: none

Theneedles canbeprepared in larger sets and stored formonths in 140mmPetri dishes, on a strip ofplasticine,inadrylocationatroomtemperature(asonpicturebelow,butwithouthumidtissue).

FillingFillneedleswiththesmallestamountofinjectionmix(1µlorless)usinganEppendorfGELoadertip(eppendorf)(althoughfilling in a drop ofmix with a regular tip at the end of theneedleworksaswell - themixmigratestotheneedletipbycapillarity,thankstotheomegadotfiber).Filledneedlesarestored inthesamePetridishasbefore,turned intoahumidchamberbytheadditionofahumid, foldedtissuecoatingtheinnersideofthelid(seepicturebelow).

Needlesareideallyfilledhoursbeforetheinjection,toallowsmallairbubbles,trappedattheneedletip,todisperse.Oneneedle isgenerally sufficient for the injectionofover100-200embryos.However,needlescanbreakorget cloggedand it’sagood idea to fill5-6needles for the injectionofa standardconstruct(400-600embryos).

Filledneedlescanbestoredat4°Candbeusedforafewdaysafterfilling.

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InjectionsetupThe injections are monitored under an inverted compound microscope equipped with a 20x

objective (e.g., Zeiss LDA-Plan20x/0,35Ph1,ZeissObjectives),bright fieldopticsanda xy-movable stage(e.g.,ZeissAxioVert.A1).

The glass needles are fitted into a needle holder (e.g., Narishige HI-7), which is controlled by amicromanipulator. The needle is also linked to an N2 compressor that facilitates injection. We use aNarishigeIM-300injector,fittedwithahandtriggerpushbutton.

Themanipulator is used for the fine-scale approach of the needle toward the embryos.We are using aLeicamechanicalmicromanipulator.Boththemicromanipulatorandthecompoundmicroscopearefixedtoaheavybase.

Thetemperatureoftheroom,whereoursetupisinstalledvariesfrom20˚Cto25˚Cwithoutaffectingtheinjectionprocess.

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Methods

Embryocollection-2to3hoursbeforeinjection,changeegg-laycapevery30-60mintoflusholdereggswithheldbyfemales.The proportion of stage 2 embryos should be above 90%. The figure below shows the posterior end ofembryoswithincreasingagebetweenstage2and3.Embryossuitableforinjectionshouldlooklikethoseatstage2on the imagesbelow (polecellsnot formed).As soonas thepolecellsare formed (stage3), theembryosaretoooldforinjection.

-Forinjection,collectanewbatchofembryosevery20minutes.

-Harvestembryos,usingamoist,medium-sizedbrush(type1or0),preferentiallyaroundtheyeastpaste.Transferthemtoacleanmeshbasket(home-madeorCostarNetwell™,Corning3477,74μmmesh).

-Washtheembryosthoroughlywithdistilledwater,usingasquirtbottle.Targetthewaterspurtdirectlytotheembryoclumpstoseparatetheembryos.

-Brieflywashtheembryoswith95%non-denaturedethanol.Theethanolpenetratesbetweenthechorionandthevitellinemembrane,makingtheembryostranslucent.This laterallowsaroughstagingunderthestereoscope.

-Wash-offtheethanolwithdistilledwater.

-Keepthemeshbasketinabitofwaterinasmall,cleanPetridish.

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Embryoalignment-Fixan18x18mmcoverslipontoamicroscopeslidebycapillaritywithadropletofwater.Transferaclutchofcleanembryosontothecoverslip,usingafine,pointy,cleanbrush(type000,alsonamed3/0).

-Startaligningtheembryoswiththemoistbrush,closetooneedgeofthecoverslip(seepicturesbelow).Theposteriorpoleoftheembryosmustpointtowardthenearestcoverslipedge. Ifpossible,arrangetheembryos in thesamedorsal-ventralorientation, ideallywith theirbackagainst thecoverslip (seebelow).Thislimitstheneedtorefocustheneedleduringtheinjection.

-Theamountofwater in thebrush iskey tomoveembryosaroundandalign themwithease.Toomuchwaterwillmaketheembryosfloataround,toolittlewater,andtheywillbehardtomoveandwillstarttogetdamages.Toadjustthedegreeofmoisture,dipthebrushtip intocleanwater(keepaPetridishwithwaternearby),andthenlightlytouchadryKimwipestissuewiththebrushtip.Thelevelofmoistureshouldbeaboutrightforaligningembryos.

-Keepthenon-alignedembryosmoistduringtheprocedure,byregularlyaddingwaterwiththebrush.

-Reject embryos older than stage 2 (i.e., make sure, pole cells have not formed; see figure above andCampos-OrtegaandHartenstein,1997).

-Let the embryo chorions dry completely (1-2 min after finishing the alignment) to ensure a firmattachmenttothecoverslip.Theembryosaredryenoughwhentherespiratoryfilamentsarewhiteandnottranslucentanymore.

-Cover the line ofembryos with oliveoil1 (prefer extravirgin organic fromCreteJ). After someseconds, the oil willhave entered thespace between thechorion and thevitelline membrane,thereby clearing theembryos.

-Movetheslidetothemicroscope stage forinjection.

1Rationaleforusingoliveoil:Drosophilaembryosareclassicallycoveredwithamixofhalocarbonoil(Spradling&Rubin,1982)forinjection.Wefoundthatthisaffectssurvivalofallstocks,mildlysoforD.melanogaster,dramaticallyforotherspecies.Unfortunatelytheviscosityofhalocarbonoilmakesitverydifficulttotakeitofftheembryosimmediatelyafterinjection.Wethereforeresortedtoanotherkindofoilthatcouldeasilyberemoved,andoliveoilcamehandy.

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Injection

Prepositioningtheneedle-Insertafilledneedleintotheneedle-holderofthemicromanipulatorandtightenthescrew.

-Orient the slide on the microscope stage with the posterior pole of the embryos toward the needle.Positiontheembryosinthelightbeam.Whilemonitoringthroughtheeyepiece,bringthefirstembryotobeinjectedtotherightsideofthefieldofview.Bringthefocustotheembryooutline.Thisfocalplanecanberecognizedbythesharpoutlineattheinterfaceofthevitellinemembraneandthechorion(seebelow).

-Manuallybringtheneedletipclosetotheembryolinewiththenakedeye(grabandmovethedistalendoftheneedleholder).

-Stillmonitoringwiththenakedeye,usethexandyknobsofthemicromanipulatortobringtheneedletipevenclosertothefirstembryooftherow.

-Monitorthenextstepsthroughtheeyepiece.Usethexandyknobstohovertheneedlebackandforthovertheembryos,untilyouseeitsshadowacrossthefieldofview.Centertheshadowinthefieldofview.Lowertheneedletipwiththezknob,untilitstipenterstheoil.

-Fine-tune thepositionof theneedle tipwithall 3 knobs to reach the final configuration (see schematicbelow).Theneedletipmustbeinthesamefocalplaneastheposterior-mostendoftheembryo.

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Openingoftheneedle-Setthepressure,suchthatafterashortpushonthemanualpressuretrigger(oursetupworkswellwith45psi),adropletofliquidisreleasedattheneedletip(plungedinoil).

-If no droplet is released, carefully bring the needle tip incontact with the chorion of an embryo (by moving themicroscopestage,nottheneedle)andmovetheembryoupanddownalongthey-axistorubtheneedletip.Dothis,whilerepeatedlypushingthepressurebutton,toseeiftheneedleopens.

-Shouldthepreviousstepproveinsufficient,attempttobreaktheneedletipopenagainsttheedgeofthecoverslip.Movethestageofthemicroscopealongthex-direction,untiltheedgeofthecoverslipisatthecenterinthefieldofview(makesuretheedgeiscoveredbyoil).Focalizeonthecoverslipedgeandbringthe needle into that focal plane by using the z knob of the micromanipulator. Carefully approach thecoverslipedge(usingthemicroscopestagecontrols)againsttheneedletip,whilerepeatedlypushingthepressurebutton.Thisisadelicateoperationthattakessomepractice;thefirstattemptswillprobablyresultinatip,toolargelyopenedandnolongersuitableforinjection.Keeptryingwithnewneedles!Theopeningishardlyvisible,butthecriterionisasuitableflowofliquidattheneedletip.

InjectionproperAstheneedleisnowopenandinthesamefocalplaneastheposterior-mostendof theembryo, injectioncanbegin.Thefollowingmovementsareperformed,usingthestageofthemicroscope:

-Insert theneedletip intotheposterior fifthof theembryo(close to the germline nuclei) in a gentlemotion. Push thepressurebuttonandinjectadropofthemix.Youwillnoticea small cloudof dyeor the turbidity (youmayoccasionallysee the embryo inflating). If you see none of this, injectagain. Back the embryo off the needle. A small amount ofliquidmayleakout.Ifthereisalwaysamilkyleak,thisisthesignofaharmfulinjectionandyoumaywanttoreplaceyourneedle.

-Itisdifficulttoestimatetheappropriateamountofmixtobeinjected.Thedashedlineinthefigureshowsthesizeofthedropinatypicalinjection.

-Center the next embryo in front of the needle. Itmay benecessary to refocalize to the outline of the new embryo,andtoadjusttheneedleposition.

-Proceedwiththe injection,skipembryosthathaveformedthe pole cells (this occurs approximately 80 minutes afterfertilization, Raff & Glover, 1989), as these will not integrate any DNA in their germline and mayunnecessarilydamagetheneedle.

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Post-injectionprocessing-Useametallicpintopokeembryosolderthanstage2underthestereoscope(tonothavetocrossthemasadults).

-Thoroughly wash off the olive oil, by pouring 95% non-denatured ethanol directly onto the embryos.Wash-offtheethanolwithdistilledwater.

-Rehydratetheembryos,byplungingthemintodistilledwaterinasmallPetridishfor10minutes(differentspecieshavedifferentdemands;asimpleH2OwashsufficesforD.melanogaster).

-Drainthewaterfromthecoverslip,byapplyingitsedgeontoacleantissue.

-Transferthecoverslipintoafoodvial(softandrichfood)andpushit,embryosup,intothefood,untiltheembryosslightlytouchit.Whenthelarvaehatch,theyareforcedtocrawlintothefood.

-Placethemarkedfoodvialintoahumidchamberat25°C,untilthelarvaehatch(24-48h).

G0crosses&F1screeningNote:totransformasmallconstruct(e.g.,anUASconstruct)injecting300-600embryosisagoodstartingpoint.Expect50-70%oftheembryostohatchintolarvae(fewerwillmakeittoadulthood).Ifthesurvivalislower,considerre-injectingimmediately.

-CollectthehatchingG0adultsandseparatethesexes.

Note: Upon hatching from the pupa, the injected animals (G0) tend to get stuck in the food and die.Transferringpupaetoafreshfoodvialincreasesthenumberofadultstobecrossedby10-20%.ThereforespraywaterontheG0pupae,toloosenthemfromthetubewall;collectthemwithabrushandstickthemtothewallofacleantube.

-SeparatelycrosseachG0maleto3virginfemalesandeachG0female(evenifnotavirgin)to2malesofastock,allowingtoseetheselectionmarkerintheprogeny.Eachcrossgetsitsownvial2.

-FormanyDrosophilaspecies,otherthanD.melanogaster,pair-matingsyieldverylittleprogeny,probablybecause there is a group effect, enhancing egg-laying. This reduces the throughput of transformantidentification considerably. Therefore, we pool G0 females by groups of 5-20, crossed to 5-10 males.Reciprocally,eachG0maleiscrossedto10-20virginfemales.

-Forseveralspecies, larvaepupate inthefoodandgetburied.Topreventthis,stickapieceofWhatmanfilterpaperintothefoodforL3larvaetopupatein.

-EfficientselectionmarkerscreeningofF1transformantsisbestdonewhen20-50adultF1havehatchedineachvial. 2G0femalescanbepooledwhentransformingaconstructwiththeɸC31system,asalltheresultinginsertionshouldbeidentical,andmixingthemdoesnotmatter.

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Troubleshooting

Problem Possiblereason(s) Solution(s)

needlesfilledwiththesameinjectionmixgetcloggedafterafewinjections

-injectionmixistooviscous-injectionmixcontainsdebris

-dilutethemixwithwater-spinthemix,transferafractionfromthetopinanewtubeàfillnewneedles

nodropcomesoutofaparticularneedle

-needleisnotopen -mechanicallyopenthetipasexplainedabove.Nottoomuch!

needlegetscloggedrepeatedly

(frompoororirregularflowtocompleteabsenceofliquidcomingout)

-embryosarenotcleanenough(coveredwithyeast)

-embryofleshenterstheneedletip

-usecleanerembryos,washbrush

-rubtheneedletipagainstthechorionofanembryo

-replaceneedle

-skipembryosthataretooold

Agoodwaytodiagnoseaconsistentproblemwithliquidflow,istofillneedleswithamixofwateranddyeandseehowtheinjectionofthissolutionbehaves.Themixshouldcomeoutoftheneedleveryeasily.

fliesdonotlayenougheggs

-theyarenotaccustomedtothecage

-yeastpasteisnotfreshenough

-cageisdirty-toofewfliesinthecage,tooold,ortooyoung

-waitanotherday,changethecapsmoreoften

-check yeast paste texture (should be liketoothpaste) and odor (should not smell likecheese)

-cleanthecagewithwater,replaceflies

embryosaretoooldupon30mincollection

-femaleswithholdtheireggs -changethecapsmoreoftenbeforestartingtoharvest,inordertoflushallwithheldeggs

embryossticktogetherduringalignment

-yeastisnotproperlyremoved -onlyharvestembryosaroundtheyeast

-washlongerandstrongerwithwater

Embryoisnotfirmlyattachedtothecoverslip

-theembryosarenotdryenoughwhenoilisadded

-increasedryingtimebeforeaddingoil

lowsurvivalrateoftheG0embryos

-notenoughrehydration

-notenoughoilremoved(choke)

-foodisnotrichenough

-theydryout

-injectionharmedthemtoomuch

Run controls to identify, at what stage thelethality mostly happens. Consider aligningembryos that you do not inject, or inject withwateronly,andprocessthemotherwiseastheinjectedones.

ManysterilefliesinG0 -germlineisdamaged -workontheshapeoftheneedles-bemoredelicateduringinjection

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ReferencesCampos-Ortega,J.A.andHartenstein,V.(1997).TheembryonicdevelopmentofDrosophilamelanogaster.Berlin;NewYork:Springer.Miller,D.F.,Holtzman,S.L.andKaufman,T.C.(2002).CustomizedmicroinjectionglasscapillaryneedlesforP-elementtransformationsinDrosophilamelanogaster.Biotechniques33,366-7,369-70,372passim.Raff,J.W.&Glover,D.M.(1989)Cell,57,611-619Spradling,A.C.andRubin,G.M.(1982).TranspositionofclonedPelementsintoDrosophilagermlinechromosomes.Science218,341-7.