Graduate Theses, Dissertations, and Problem Reports 2003 Gene expression profiling in prepubertal and adult male mice using cDNA and oligonucleotide microarrays Lisa Marie Tomascik-Cheeseman West Virginia University Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Tomascik-Cheeseman, Lisa Marie, "Gene expression profiling in prepubertal and adult male mice using cDNA and oligonucleotide microarrays" (2003). Graduate Theses, Dissertations, and Problem Reports. 1844. https://researchrepository.wvu.edu/etd/1844 This Dissertation is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. 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GENEEXPRESSIONPROFILINGIN PREPUBERTALANDADULTMALEMICEUSING CDNAANDOLIGONUCLEOTIDEMICROARRAYS LisaMarieTomascik-Cheeseman Dissertationsubmittedtothe GeneticsandDevelopmentalBiologyProgram, CollegeofAgriculture,Forestry,andConsumerSciences, atWestVirginiaUniversity inpartialfulfillmentoftherequirements forthedegreeof DoctorofPhilosophy in Genetics JoginderNath,Ph.D.,Co-Chair AndrewJ.Wyrobek,Ph.D.,Co-Chair WalterKaczmarczyk,Ph.D. DanielPanaccione,Ph.D. SharonWenger,Ph.D. Morgantown,WestVirginia 2003 Variations in gene expression are the basis of differences in cell and tissue function, response to DNA damaging agents, susceptibility to genetic disease, and cellular differentiation.Thepurposeofthisdissertationresearchwastocharacterizevariationin basal gene expression among adult mouse tissues for selected stress response, DNA repair and damage control genes and to utilize variation in temporal gene expression patternstoidentifycandidategenesassociatedwithgermcelldifferentiationfrommitosis through meiosis in the prepubertal mouse testis. To accomplish these goals, high throughputanalysesofgeneexpressionwereperformedusingcustomcDNAandrandom oligonucleotidemicroarrays.cDNAmicroarraytechnologywasoptimizedbyevaluating theeffectsofmultiplehybridizationandimageanalysismethodologiesonthemagnitude of background-subtracted hybridization signal intensities. The results showed that hybridizinglowerprobequantitiesinabufferdevelopedatLawrenceLivermoreNational Laboratorytotryptone-blockedmicroarraysimprovedsignalintensities.Inaddition,the error in expression ratio measurements was significantly reduced when microarray images were preprocessed. A custom cDNA microarray comprised of 417 genes and enriched for stress response, DNA repair, and damage control genes was used to investigate basal gene expression differences among adult mouse testis, brain, liver, spleen, and heart. Genes with functions related to stress response exhibited the most variation in expression among tissues whereas DNA repair-associated gene expression variedtheleast.Randomoligonucleotidemicroarrayscomprisedof~10,000geneswere usedtoprofilechangesingeneexpressionduringthefirstwaveofspermatogenesisinthe prepubertalmousetestis.Approximately550genesweredifferentiallyexpressedasmale germcellsdifferentiatedfromspermatogoniatoprimaryspermatocytes.Thesefindings suggestthatthe313unannotatedsequencesand178geneswithknownfunctionsinother biological pathways have spermatogenesis-associated roles. This dissertation research showedthatmicroarraysareausefultoolforquantitatingtheexpressionoflargenumbers ofgenesinparallelundernormalphysiologicalconditionsandduringdifferentiation.It hasalsoprovidedcandidategenesforfutureinvestigationsofthemolecularmechanisms underlying(1)tissue-specificDNAdamage responseand geneticdiseasesusceptibility and(2)cellulardifferentiationduringtheonsetandprogressionofspermatogenesis. Thisdissertationisdedicatedtomyhusband,Matthew, forhisadvice,encouragement,and(mostofall)love. Thankyouforgivingmethecouragetodream. Thisworkisalsodedicatedtomyparents,ThomasandCarol Tomascik, andtomygrandmother,SophieDuzen inhonoroftheirtirelessloveandsupport. Inmemoryofmygrandfather,AndrewDuzen. iv TableofContents Abstract ii Acknowledgementsvii ListofTables viii ListofFigures x ListofAbbreviations xii Chapter1. Introduction 1 DissertationObjectives 4 Chapter2. ExpressionMicroarrayTechnology ChapterOverview 10 Section2.1 Fluorescentexpressionmicroarraytechnologyreview 2.1.1 Introduction 11 2.1.2 TypesofExpressionMicroarrays 13 2.1.3 ProbeGeneration,Hybridization,andImageCapture 14 2.1.4 MicroarrayDataAcquisitionandAnalysis 20 2.1.5 Summary 21 2.1.6 References 22 Section2.2 Optimizationoftwo-colorfluorescencecDNA microarrayhybridizationsvisualizedwithawhite lightimagingsystem 2.2.1 Abstract 27 2.2.2 Introduction 27 2.2.3 MaterialsandMethods 31 2.2.4 Results 37 2.2.5 Discussion 46 2.2.6 References 50 Section2.3 Accuratequantitationoffluorescencemicroarrays 2.3.1 Abstract 51 2.3.2 Introduction 51 2.3.3 MaterialsandMethods 53 2.3.4 ImageProcessingandAnalysisMethods 58 2.3.5 Results 69 2.3.6 Conclusions 73 2.3.7 Auspices 74 2.3.8 References 75 v Chapter3. DifferentialBasalExpressionofGenesAssociated withStressResponse,DamageControl,andDNA RepairAmongMouseTissues 3.1 Abstract 77 3.2 Introduction 78 3.3 MaterialsandMethods 80 3.4 Results 87 3.5 Discussion 100 3.6 Acknowledgements 106 3.7 References 107 Chapter4. MitoticandMeioticGeneExpressionProfilingof MaleGermCells 4.1 Abstract 114 4.2 Introduction 115 4.3 MaterialsandMethods 117 4.4 Results 121 4.5 Discussion 133 4.6 Acknowledgements 137 4.7 References 138 Chapter5. Conclusions 152 Appendices A Symbols,names,biologicalpathways,I.M.A.G.E. cloneIDs,andcDNAmicroarraydatafor152 geneswithdifferentialexpression amonghealthyadultmousetissues 155 B Allannotatedgenesshowingsignificantdifferences inexpressionduringthetransitionfrommitosisto meiosisintheprepubertalmousetestis 160 C Unannotatedsequenceswithsignificantexpression differencesduringthefirstwaveofspermatogenesis intheprepubertalmouse 167 D Functionalgenomics-relatedwebsites 173 E CurriculumVitae 175 F Publishedresearcharticles 180 vi Acknowledgements Dr. Joginder Nath, Academic Advisor and graduate committee Co-chair: Words can’texpresshowthankfulIamtohavebeenyourstudent.Yourguidancehasenabled metogrowbothpersonallyandprofessionally. Dr.AndrewJ.Wyrobek,ResearchAdvisorandgraduatecommitteeCo-chair:Iam so grateful for the opportunity to be a member of your laboratory. Thank you for constantlychallengingmeandalwaysfindingtimeformyresearch. Dr.WalterKaczmarczyk,Dr.DanielPanaccione,andDr.SharonWenger,graduate committeemembers:Thankyouforreviewingthedissertationandprovidingvaluable comments. Past and present members of the Wyrobek laboratory: Thank you for the spirited scientificconversationsandcamaraderie.Iwouldespeciallyliketorecognize: Dr. Francesco Marchetti: I could never thank you enough for everything you havetaughtme.Iwillalwayscherishourfriendship. Eddie Sloter: I have enjoyed every discussion (scientific and otherwise). You havebeenawonderfulfriend,andIcannotimaginewhatthisexperiencewould havebeenlikewithoutyou. Dr.MatthewA.Coleman:Thanksforalwayskeepingmeontaskbyasking,“Is thatdissertationfinished yet???”Iadmire yourscientificinsightandappreciate yourfriendship. FrancescaHill:Thanksforsharingyourcytogeneticexpertiseandfriendship. Nancy Wrigley: Thank you for the administrative assistance and motherly advice. Dr.DavidO.NelsonandSheaGardner:Iamtrulythankfulforallofyourassistance withthemicroarraystatisticalanalyses. LauraMascioKegelmeyer:ThanksforpatientlyteachingmeeverythingIeverneeded toknowaboutUNIXsystems.Perhapsmoreimportantly,thankyoufortheafternoontea. TheTomascikandCheesemanfamilies:Yourloveandunwaveringsupporthasmade thisdreampossible.IpromisetogetaREALjob…someday. Dr.SaraFrias,AnaClaudiaVelazquez-Wong,DougandLindaHamilton,andthe Jackson family: I will never forget the laughter, smiles, and many words of encouragement.Thankyouforyourfriendship. vii ListofTables Chapter2. Page ExpressionMicroarrayTechnology Section2.2 OptimizationofcDNAmicroarrayhybridizationsvisualized withaXenonlightsource 2.2.1 PrimersusedtoamplifycDNAclonesforthegene-specific hybridizationexperiments 33 2.2.2 Experimentaldesignforthegene-specificandtissuesample hybridizations 35 2.2.3 Comparisonofbackground-subtractedsignalintensities obtainedfromgene-specifichybridizations 40 2.2.4 CharacterizationofthesignalintensitiesobtainedforeachAlexa Fluorfollowingdifferenttissuesamplehybridizationprotocols 43 2.2.5 Relationshipbetweentissuesamplehybridizationprotocoland numberofgeneswithsignalintensityrati≥os1.5 45 Section2.3 Accuratequantitationoffluorescencemicroarrays 2.3.1 Sixgeneswereusedtobuildagroundtruthseriesoverten microarrayslides 56 2.3.2 Errormeasurementsobtainedforthegroundtruthseries followingdifferentimageanalysismethods 71 Chapter3. DifferentialBasalExpressionofGenesAssociatedwithStressResponse, DamageControl,andDNARepairAmongMouseTissues 3.1 Differencesinthegeneexpressionratiosfromreplicate independenthybridizations 91 3.2 DistributionofFratiosfordifferentiallyexpressedgeneswith respecttobiologicalpathway 96 3.3 Differentiallyexpressedstressresponse,DNArepair,and