Blastocystembryotransferintopseudopregnantrecipientfemalemouse

TheRecipient

Carefulselectionoftherecipientismostimportantasthepupsaretheendresultofalotofhardwork.IpersonallyuseSwissWebsterorCD-1mice,astheyarequietandmakeexcellentmothers,althoughdobecomeoverweightquicklyandexhibitbadplanesofanesthesiawhenheavy.Thisisalsoaveryinexpensivemousetouse.Asanalternate,anotherstrainIhaveusedwithconsiderablesuccessisB6D2F1.Thesemicearehardyanddisplayhybridvigor.

TheEquipment

Forsurgery,Iclean(with70%alcohol)thefollowing(allRobozinstruments):

5pairsofforceps

1pairPattern3c1pairstraightserrated,3.5"1paircurvedserrated,3.5"

1pairof3.5"sharp/sharpscissors(Surecut)

25guagehypodermicneedle

Autoclipmetalwoundclipper

1serafinclip

1mouthPipetterandhand-pulledtransferpipette

TheAnesthetic

Avertin(2,2,2tribromoethanol)isfoundtobequiteeffective.Formethodofpreparation,see"ManipulatingtheMouseEmbryo",CSHLPress,ISBN0-87969-384-3.Storewrappedintinfoilat4^oCasthisreagentislightsensitive.Testaftermakinganewbatch.Shakewellbeforeuse.

TheTransfer

Selectamousethathasbeenpluggedbyavasectomizedmale2.5dayspreviously.Donotuseanmousethatappearslighterthan25g,asunderweightmicetendtore-absorbtheembryosastheyarenotphysicallyreadytosupportapregnancy.Overweightmicecanmakesurgerydifficultbytheabsorptionofanaestheticintothefatreducingthepotencyoftheanesthetic;also,thepresenceoffatmeansthepresenceofbloodvessels,andcuttingthroughalltheextrafatcausesalotofunnecessarybleeding.Thismakesitdifficulttoseewhatyouaredoingandmayalsoclogupthetipofyourtransferpipette.

AnesthetisemousewithAvertin,admiNISTeredintraperitoneally.Afteradministeringtheanesthetic,putthemousebackintothecagefromwhichitcame.Themousewillbemorerelaxedwhenplacedinafamiliarenvironmentandtheanestheticwillactmorequicklythanitwouldonadistressedmouse.

Tocheckthatthemouseisfullyanesthetised,pressorsqueezethepadsofthefeet.Ifthemousecanfeelthisitwilltrytowithdrawitslegfromyourgrasp(Pedalreflex).Donotcommencesurgeryuntilthereisnoreflexreactiontothistest.

Taketheanesthetisedmouseandlayitonit"sbellyonapetridishlid,takingcaretokeeptheairwayclearbyrestingtheteethontheedgeofthepetridish.Thismakesiteasiertomovethemousearoundwithouthavingtoactuallytouchit.Swabtheincisionareawith70%ethanol.

TheSurgery

UsethepairofSurecutscissorsandonepairofserrated,curvedforcepsforcuttingtheskin.Theincisionshouldbemadeapproximately0.5cmawayfromthemidlineandbetweenthenaturalhumpofthebackandthepointwheretherearlegjoinstheaBDomen.Usinga(lint-free)tissuedampenedwith70%ethanol,carefullywipetheincisionsite,sweepingawaythecuthair.Wipethescissorswitha70%ethanoldampenedtisuetoremoveanyhair.Graspingonesideoftheincisionatatime,carefullyintroducethebladesofthescissors(whileclosed)betweentheinsideofthepeltandthebodywallforapproximately1cm.Openandclosethebladestocleartheconnectivetissueinthisarea.Movetheskinarounduntilthenerve(awhiteline,usuallyseenwithassociatedcapillary)canbeseenrunningacrossthebodywall.Thelightcoloroftheovarianfatpadcanbeseenunderthebodywallinthisarea.Usingapairofpattern3cforceps,pinchthebodywallandnicktogiveanincision0.5cmacross.Reachinwiththestraightserratedforceps(whileholdingthebodywallwithanotherpairofcurvedforceps)andgrasptheovarianfatpad,andremovetotheexteriorwhereitcanbeanchoredusingaserafinclip.

Resttheserafinclipacrossthemouse"sbacktoholdtheuterusinplace.Iftheuterusoruterinehorncontinuallyslipbackintothecavityitmaybenecessarytogentlyliethemouseonthesidebeingcarefulnottoblocktheairway.Thetransferpipetteshouldnowbeloaded.Aminimumof9-10embryostotalmustbetransferred,anylessthanthisandthechancesofapregnancyresultingareseverelyreduced.Embryosmaybeimplantedinbothuterinehornsorunilaterally.Thetransferpipettecanbeloadedanumberofdifferentways;thefollowingisonepopularmethod.TakeupanamountofHepesbufferedmediuminthetipofthetransferpipette,thenmakeasmallbubblebytakingupalittleair.Thentakeupsomemoremedium-roughlythesamevolumeastheairbubble,thenanotherairbubble,samesizeasbefore.Thentakeupabout2-3cmofbufferedmediaandthenatinyairbubbleoncemore.TakeyourembryosinthesmallestpossIBLevolumeofmedium,liningthemupsidebysideinthetransferpipette.Introduceanothertinyairbubblewhenalltheeggsareloaded.Somepeopleusemineraloilinsteadoforinconjunctionwithairbubblesforpressurecontrol.Thisisaperfectlyacceptablepractice;howevercaremustbetakentoavoidintroducingmineraloilintotheoviductsasthiscandramaticallyreducelittersizebyinterferingwiththeciliary-driveneggtransportinthisstructure,aswellaspredisposingtheanimaltouterineinfection.

Oncethepipetteisloadedandtheuteruspositioned,movethepetridishlidsupportingthemousetothemicroscopeandturnontheoverheadlightsource.Oncethelightsandfocushavebeenadjustedandthemousepositionedtosuityourself,graspthetopoftheuterinehornattheedgeandmakeaholeusinga25guagehypodermicneedlethroughintothelumen.ThesiteofthisholeshouldbenearthelastloopoftheoviductwhereitdescendsintothetopoftheuterinehornattheUTjunction.

Introducingtheembryosatthissitewillplacethematthecorrectpositiondevelopmentally.Theembryosinthetransferpipettearethreadedthroughtheholeintheuterusintothelumenandexpelled.Bywatchingtheprogressofthelargerairbubblesusedtoprovidepressurecontrolitcanbedeterminedwhentheembryoshavebeenplacedinsidethelumen.

Withthetransfercomplete,theserafinclipcannowberemovedandtheuterusgentlyeasedbackintothebody.Donottouchtheuterus,buteaseitbackbytheedgesoftheincisioninthebodywallandallowingtheuterustofallbackin,withoutactuallyhandlingit.Thisprocedureisthenrepeatedontheotheruterinehornifdoingabilateraltransfer.Theincisioninthebodywallisnotsutured.TheskinisclosedwithAutoclips-twoperincisionisusuallysufficient.Autoclipwoundclipsareusedontheskininsteadofsutureasthemicefrequentlywillchewatthesuturethreadandeffectivelyopentheirwounds.

Oncesurgeryiscomplete,themouseisplacedinaboxofcleanautoclavedsawdust.Underanesthetic,mammalsareunabletoretainheataseffectivelyaswhenconscious.Forthisreason,themouseshouldbewrappedinatissuetohelpkeepitwarm.Useofaheatingpadorevenindirectheatfromaslidewarmercanbeusedtocarefortheanimalpost-operativelyuntilitregainsconciousness.Allanimalsshouldhaverecoveredsufficientlyfromanestheticbeforebeingreturnedtotheanimalroomandleftunattended.Recipientmiceshouldbehandledwithcareaspregnantmicebecomeeasilystressed,sometimesleADIngtoabortion,orevencannibalismofpups.

Ifallgoeswellandapregnancyresults,thepupsshouldbebornapproximately17daysafterthetransfer,dependentuponstrain.