Characterization of A-transgenic mouse expressing the HSV-1 latency associated transcript

2004·4.9 MB·English

by  Gussow, Anne M

CHARACTERIZATIONOFATRANSGENICMOUSEEXPRESSINGTHEHSV-1
LATENCYASSOCIATEDTRANSCRIPT
By
ANNEM.GUSSOW
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNrVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
2004
Copyright2004
by
ANNEM.GUSSOW
Tomyhusband,Karl,whoismybestcheerleader,
andmyson,Seth,whoistooyoungtounderstand.
ACKNOWLEDGMENTS
Iwouldfirstliketothankmymentor,Dr.DavidBloom,forhisguidanceand
directioninthecompletionofthisworkandforprovidingmewiththeopportunityto
completemydoctoralstudiesattheUniversityofFlorida. Iwouldalsoliketothankmy
committee,Dr.RichardCondit,Dr.SueMoyer,andDr.PaulReier,fortheiruseful
discussionsandsuggestionstothisproject.
IwouldliketothankDr.RobertBonneauforencouragingmetoapplytograduate
schoolandforgivingmeastartinmyresearchcareer,andDr.EddieCastanedafor
helpingtoeasemytransitionfromArizonaStatetotheUniversityofFlorida.
Iwouldliketoexpressmygratitudetothepeoplewhohaveworkedwithmein
Dave'slabovertheyears,boththeASUgroup,RickJarman,RobertTran,JerryO'Neil,
NikiKubat,MelaniePaquette,andLeeGary,aswellastheUFgroup,TonyAmelio,
ZaneZeier,NicoleGiordiani,PeterjonMcAnanyandLorettaArrue. Eachofthemhas
broughtaspecialqualitytothelabthatwillnotbeforgotten.
Thebiggestacknowledgmentgoestomyhusband,Karl,andmyson,Seth.
Withouttheloveandsupportofbothofthemthisgoalwouldhavefallenshortalong
timeago.IthankKarlforthe2500milesof1-10,1hopethejourneyhasbeenworththe
sacrificesthathehasmadeasaresult. AlthoughSethistooyoungtounderstandanyof
this,cominghometohissimpleoutlookonlifehasputthingsintoperspectiveonmany
occasions.
IV
Ialsowanttothankmymotherwhohaslistenedtotheupsanddownsallalongthe
wayandalwaysbeensupportiveofmenomatterwhat. It'sbeenalongjourneyaround
thecountryandI'mfinallygoingto"settledown"inoneplace. Tomybrothers,Marty
andKarl,whohaven'tunderstoodallofwhatmyresearchisabout,thanksforlistening
andpretendingtounderstand.ToCathyKostick,whohasbeenlikeasistertome,thank
youforalwaysbeingtherethroughtheyears,ingoodtimesandinbad,yourfriendship
meansmoretomethanIcanexpress.
Lastbutcertainlynotleast,Iwouldliketothankmyfriendswhohavebeenthere
formeeverytimethatIwasreadytogiveup;thoseofyouwhoremindedmetodothe
nextrightthing,takeitonedayatatimeandputfirstthingsfirst. Innoparticularorder:
Corliss,Aileen,Julie,Betty,Sheila,Eve,Heather,Leona,MarthaAnn,Jennifer,Sarah,
Claire,Alice,Polly,Joan,MaryEllen,Marty,Ted,Steve,Dale,Rosemary,Julia,Casey,
Diedre,Buster,BJ,Warren,Jim,Walt,Dan,andJoe;youareeachaspecialpartofmy
lifeandofmakingthisdreamcometrue. AndtowhomeverIhaveforgotten,youknow
whoyouare,andIthankyou.
TABLEOFCONTENTS
Page
ACKNOWLEDGMENTS iv
LISTOFTABLES viii
LISTOFFIGURES ix
KEYTOSYMBOLS xi
ABSTRACT xiv
CHAPTER
1 INTRODUCTION 1
HSVClinicalFeatures 1
ImmuneResponsetoHSV 2
GeneralHSVCharacteristics 3
HSVGeneRegulation 6
AnimalModelsofHSVLatencyandReactivation 11
FunctionofLAT 13
UseofTransgenicstoStudyDisease 18
HypothesestobeTestedUsingTransgenicApproach 20
2 GENERATIONOFTRANSGENICMOUSEEXPRESSINGAPORTIONOF
THEHSV-1LATENCYASSOCIATEDTRANSCRIPT 23
Overview 23
MaterialsandMethods 24
Results 33
Discussion 41
3 EXPRESSIONPROFILEOFTHELATTRANSGENE 43
Overview 43
MaterialsandMethods 44
Results 51
Discussion 67
VI
4 CHARACTERIZATIONOFTHEEXPRESSIONOFLATINTRANSONTHE
COURSEOFHSV-1INFECTIONINMICE 73
Overview 73
MaterialsandMethods 74
Results 78
Discussion 85
5 OVERALLCONCLUSIONS 88
APPENDIX
A MAPANDSEQUENCEOFTHEpLAT/LATPLASMID 91
B PCRPRIMERSEQUENCES 98
C EXPRESSIONOFTHELATTRANSGENEISNOTAGEDEPENDENT 100
LISTOFREFERENCES 104
BIOGRAPHICALSKETCH 112
vn
LISTOFTABLES
Table E^ge
2-1 Determinationoftransgeniccopynumberbyreal-timePCR 35
3-1 QuantitationofLATExpressingCellsinDorsalRootGangliabyinsitu
hybridizationfortheLAT5'exon 63
B-l ConventionalPCRPrimersandLocations 98
B-2 RealtimePCRPrimerandProbeSequences 99
vni
LISTOFFIGURES
Figure Eage
1-1 DiagramoftheHSV-1virion 4
1-2 DiagramoftheHSV-1genome 5
1-3 RegulationofthedifferentHSVgenepromoterclasses 7
1-4 DiagramofthefunctionofHSV-1LAT 13
2-1 DiagramoftheLATTransgeneInsert 26
2-2 HSVtransgenecopynumberdeterminationbyslotblothybridizationanalysis 34
2-3 MappingoftheLATtransgeneintheLATtransgenicmouse 37
2-4 PCRmappingthetransgeneinsert 38
2-5 ExpressionoftheLATtransgene 40
3-1 ExpressionoftheLATtransgenepergramoftissue 53
3-2 ExpressionoftheLATtransgenenormalizedto18sRNA 55
3-3 LATtransgeneexpressionisnotagedependentintissuestypicallyinvolvedinthe
HSVinfectionwhennormalizedonaperweightbasis 58
3-4 ExpressionoftheLATtransgeneisnotagedependentintissuesinvolvedinthe
HSVinfecctionwhencalculatedpercellbynormalizingto18sRNA 58
3-5 Insituhybridizationofalatentlyinfecteddorsalrootganglia 59
3-6 Insituhybridizationfor5'LATexon 61
3-7 InsituhybridizationforLATintronintransgenicDRG 62
3-8 InsituhybridizationforLAT5'exoninbrain 64
3-9 InsituhybridizationofthetransgenicbrainwithLATintronprobe 65
3-10 Insituhybridizationinthetransgenicspinalcord 66
IX
3-11 Insituhybridizationoftransgenicfoot 68
3-12 Insituhybridizationinthetransgenickidney 69
4-1 Relativeamountsofinfectiousvirusinthefeetoftransgenicandnon-transgenic
miceinfectedwithHSV-1strain17+duringacutetimespostinfection 79
4-2 TiterofinfectiousvirusinDRGoftransgenicandnon-transgenicmiceinfected
withHSV-1strain17+atacutetimespostinfection 80
4-3 QuantitationofHSV-1genomespresentinmiceinfectedwithHSV-117+atlatent
timespostinfection °1
4-4 ReativationofHSV-1fromtransgenicandnon-transgenicmicebyexplantco-
cultivationoflatentlyinfectedDRG 83
4-5 DiagramoftheLATregionofHSVillustratingthelocationofthe17A480virus
andtheLATtransgeneinrelationtothevirulencefunctionofLAT 84
4-6 Relativeamountsofinfectiousvirusinfeetoftransgenicandnon-transgenicmice
infectedwithadeletionmutantofHSV-1atacutetimespostinfection 84
4-7 RelativeamountsofinfectiousvirusinDRGoftransgenicandnon-transgenicmice
infectedwithadeletionmutantofHSV-1atacutetimespostinfection 85
A-l PlasmidmapoftheHSV-1LATtransgenicconstruct 91
C-l ExpressionoftheLATtransgeneinneuraltissues 101
C-2 ExpressionoftheLATtransgeneinnon-neuraltissues 101
C-3 ExpressionoftheLATtransgeneinneuraltissuesrepresentedascopiesper18s
RNA 102
C-4 ExpressionoftheLATtransgeneinnon-neuraltissuesrepresentedascopiesper
18sRNA 103