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Methods in Molecular Biology 1463 Michael Buszczak Editor Germline Stem Cells Second Edition M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire,AL109AB,UK Forfurther volumes: http://www.springer.com/series/7651 Germline Stem Cells Second Edition Edited by Michael Buszczak Department of Molecular Biology University of Texas Southwestern Medical Center Dallas, TX, USA Editor MichaelBuszczak DepartmentofMolecularBiology UniversityofTexasSouthwesternMedicalCenter Dallas,TX,USA ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-4939-4015-8 ISBN978-1-4939-4017-2 (eBook) DOI10.1007/978-1-4939-4017-2 LibraryofCongressControlNumber:2016945156 ©SpringerScience+BusinessMediaNewYork2008,2017 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthorsandtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeenmade. Printedonacid-freepaper ThisHumanaPressimprintispublishedbySpringerNature TheregisteredcompanyisSpringerScience+BusinessMediaLLCNewYork Preface Adult stem cells maintain tissue homeostasis by producing progeny that replace cells lost throughthecourseofnormalcellularturnoverorbecauseofinjury.Germlinestemcellsare uniqueamongst stem cellsbecause theyproduce daughtercellsthat develop into gametes, which ultimately serve to give rise to the next generation. Thus, germline stem cells are essentialfor thecontinuedpropagationofmanysexuallyreproducingorganisms. Germcellsareimbuedwithseveraluniqueproperties.Theyaretheonlycellstoundergo meiosis. Germ cellsacross species express many of thesame markersand experienceexten- siveepigeneticreprogramming.Thesecellshavespecializedmechanismsthathelpmaintain the integrity of the genome and prevent the integration of selfish DNA elements. Several somatictumorsexpressgenesnormallyspecifictogermcellsandgermcellsthemselvescan giverisetospecifictypesofcancer.Therefore,abetterunderstandingofgermcellswillhave abroadimpactacrossmanyfieldsofbiology. Germline stem cell biology has witnessed an explosion of new findings and techniques since that last edition of Germline Stem Cells in the Methods in Molecular Biology series. The optimization of live-cell imaging techniques, improved cell purification protocols and theidentificationofgermcellsinanumberofgeneticallytractableorganismsnowallowsfor the characterization of germ cells at a greater depth and higher resolution than previously attainable.Thisnew editionofGermlineStemCells isintended toprovideresearcherswith selected genetic, molecular, biochemical, and cell biological techniques used in germ cell research.Whilethefocushereisonprimordialgermcellsandgermlinestemcells,manyof the techniques and principles presented in the chapters of this issue may be applicable to manydifferenttypesofadultstemcells. IwouldliketothankProf.JohnM.WalkerandthestaffatSpringerfortheirinvitation, assistance, and patience during the preparation of this book. I would like to thank Nevine Shalabyforherhelpthroughoutthisentireprocess.Iwouldalsoliketoexpressmysincere appreciationandgratitudetothevariouscontributorsforsharingtheirinsightsandexpertise withthegermlinestemcellresearchcommunity. Dallas,TX,USA MichaelBuszczak v Contents Preface ..................................................................... v Contributors................................................................. ix 1 AnalysisoftheC.elegansGermlineStemCellPool.......................... 1 SarahL.Crittenden,HannahS.Seidel,andJudithKimble 2 MethodsforStudyingtheGermlineoftheHumanParasite Schistosomamansoni..................................................... 35 JulieN.R.CollinsandJamesJ.CollinsIII 3 EvaluationoftheAsymmetricDivisionofDrosophila MaleGermlineStemCells ............................................... 49 MayuInabaandYukikoM.Yamashita 4 LiveImagingoftheDrosophilaTestisStemCellNiche ...................... 63 LeahJ.GreenspanandErikaL.Matunis 5 RNAIsolationfromEarlyDrosophilaLarvalOvaries ........................ 75 DanaGanczandLilachGilboa 6 Live-CellImagingoftheAdultDrosophilaOvaryUsing ConfocalMicroscopy.................................................... 85 NevineA.ShalabyandMichaelBuszczak 7 MeasurementofmRNAPoly(A)TailLengthsinDrosophila FemaleGermCellsandGerm-LineStemCells ............................. 93 AymericChartier,WillyJoly,andMartineSimonelig 8 IdentificationofGerm-LineStemCellsinZebrafish......................... 103 BruceW.Draper 9 PrimordialGermCellIsolationfromXenopuslaevisEmbryos ................ 115 AmandaM.Butler,TristanAguero,KarenM.Newman, andMaryLouKing 10 Single-CellLineageAnalysisofOogenesisinMice.......................... 125 LeiLeiandAllanC.Spradling 11 VisualizationandLineageTracingofPax7+Spermatogonial StemCellsintheMouse................................................. 139 GinaM.Aloisio,IleanaCuevas,YujiNakada,ChristopherG.Penã, andDiegoH.Castrillon 12 TransplantationasaQuantitativeAssaytoStudyMammalian MaleGermlineStemCells ............................................... 155 AileenR.HelselandJonM.Oatley 13 MouseFetalGermCellIsolationandCultureTechniques ................... 173 CassyM.Spiller,GuillaumeBurnet,andJosephineBowles vii viii Contents 14 RattusnorvegicusSpermatogenesisColony-Forming Assays.................. 185 F.KentHamra 15 IdentificationofMousepiRNAPathwayComponents UsingAnti-MIWI2Antibodies........................................... 205 TakamasaHirano,HidetoshiHasuwa,andHaruhikoSiomi 16 EfficientInductionandIsolationofHumanPrimordial GermCell-LikeCellsfromCompetentHuman PluripotentStemCells................................................... 217 NaokoIrieandM.AzimSurani Index ...................................................................... 227 Contributors TRISTANAGUERO (cid:1) DepartmentofCellBiology,UniversityofMiamiMillerSchoolof Medicine,Miami,FL,USA GINAM.ALOISIO (cid:1) DepartmentofPathologyandCecilH.andIdaGreenCenter for ReproductiveBiologySciences,UniversityofTexasSouthwesternMedicalCenter,Dallas, TX,USA JOSEPHINEBOWLES (cid:1) SchoolofBiomedicalSciences,TheUniversityofQueensland,Brisbane, QLD,Australia GUILLAUMEBURNET (cid:1) SchoolofBiomedicalSciences,TheUniversityofQueensland,Brisbane, QLD,Australia MICHAELBUSZCZAK (cid:1) DepartmentofMolecularBiology,UniversityofTexasSouthwestern MedicalCenter,Dallas,TX,USA AMANDAM.BUTLER (cid:1) DepartmentofCellBiology,UniversityofMiamiMillerSchoolof Medicine,Miami,FL,USA DIEGOH.CASTRILLON (cid:1) DepartmentofPathologyandCecilH.andIdaGreenCenter for ReproductiveBiologySciences,UniversityofTexasSouthwesternMedicalCenter,Dallas, TX,USA AYMERICCHARTIER (cid:1) mRNARegulationandDevelopment,InstitutdeGeńe´tiqueHumaine, CNRSUPR1142andUniversityofMontpellier,Montpellier,Cedex5,France JULIEN.R.COLLINS (cid:1) DepartmentofPharmacology,UniversityofTexasSouthwestern MedicalCenterr,Dallas,TX,USA JAMESJ.COLLINSIII (cid:1) DepartmentofPharmacology,UniversityofTexasSouthwestern MedicalCenter,Dallas,TX,USA SARAH L.CRITTENDEN (cid:1) DepartmentofBiochemistry,HowardHughesMedicalInstitute, UniversityofWisconsin-Madison,Madison,WI,USA ILEANA CUEVAS (cid:1) DepartmentofPathologyandCecilH.andIdaGreenCenter for ReproductiveBiologySciences,UniversityofTexasSouthwesternMedicalCenter,Dallas, TX,USA BRUCEW.DRAPER (cid:1) DepartmentofMolecularandCellularBiology,Universityof California,Davis,Davis,CA,USA DANAGANCZ (cid:1) DepartmentofBiologicalRegulation,WeizmannInstituteofScience, Rehovot,Israel LILACH GILBOA (cid:1) DepartmentofBiologicalRegulation,WeizmannInstituteofScience, Rehovot,Israel LEAHJ.GREENSPAN (cid:1) DepartmentofCellBiology,JohnsHopkinsUniversitySchoolof Medicine,Baltimore,MD,USA F.KENTHAMRA (cid:1) DepartmentofPharmacology,CecilH.&IdaGreenCenter for ReproductiveBiologySciences,UniversityofTexasSouthwesternMedicalCenter,Dallas, TX,USA HIDETOSHIHASUWA (cid:1) DepartmentofMolecularBiology,KeioUniversitySchoolofMedicine, Tokyo,Japan AILEEN R.HELSEL (cid:1) Center forReproductiveBiology,SchoolofMolecularBiosciences, CollegeofVeterinaryMedicine,WashingtonStateUniversity,Pullman,WA,USA ix