Auditory and Vestibular Research: Methods and Protocols

416 Pages·2009·6.85 MB·English

by  Casilda Trujillo-Provencio, TuShun R. Powers, David R. Sultemeier, Elba E. Serrano (auth.), Bernd Sokolowski (eds.)

Auditory and Vestibular Research
M E T H O D S I N M O L E C U L A R B I O L O G YTM
John M. Walker, SERIESEDITOR
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METHODS IN MOLECULAR BIOLOGY TM
Auditory and Vestibular Research
MethodsandProtocols
Editedby
Bernd Sokolowski,
PhD
UniversityofSouthFlorida,CollegeofMedicine,Tampa,FL,USA
Editor
BerndSokolowski
CollegeofMedicine
UniversityofSouthFlorida
Tampa,FL
USA
[email protected]
SeriesEditor
JohnM.Walker
SchoolofLifeSciences
UniversityofHertfordshire
Hatfield,Hertfordshire,AL109AB,UK
ISBN:978-1-934115-62-6 e-ISBN:978-1-59745-523-7
ISSN:1064-3745 e-ISSN:1940-6029
DOI10.1007/978-1-59745-523-7
LibraryofCongressControlNumber:2008934544
(cid:2)c 2009HumanaPress,apartofSpringerScience+BusinessMedia,LLC
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Preface
Hearing is a sensory modality critical to both language and cognitive development.
In its absence, and without sensory input through another modality, such as the
manual/visual modality of sign language, cognitive and language development can
be severely impaired in the earliest formative years of a child. In its endeavor to dis-
cover the mechanisms underlying audition, the field of auditory science has provided
richcomparativephysiologicalstudies,allowinginsightsintoboththemicromechani-
cal and electrochemical world of this system. For many years, the auditory/vestibular
sciences have been influenced by the discoveries of electrical engineers and sensory
physiologists, who have provided insights into the functions of this dynamic system.
The early discoveries in these fields, as well as advancements in microprocessing and
materials technologies, provided a means whereby hearing could be regained partly
throughtheuseofabionicdevice,knownasacochlearimplant.Presently,thisdevice
and the auditory brainstem implant are the only ones to prosthetically replace brain
function.
With the advent of molecular biology tools, such as RT-PCR, the auditory and
vestibular fields have made great strides in understanding the genetic basis for various
hearingandbalancedisordersoverthepastfifteentotwentyyears.Thesetechnologies
permittedthediscoveryofgenesthatcontrolinnerearstructureandfunctionbyover-
coming the hurdle of working with small amounts of tissue, as found in the inner ear.
The amplification of genes with RT-PCR provided a means to discover gene expres-
sion in the small, inner ear endorgans during development, as well as in damaged
and normal sensory epithelia in the adult. The use of gene knockout animal models
provided the means to verify the effects of genes critical to the development of this
system, whereas in situ hybridization localized newly discovered gene transcripts. As
these technologies continue to broaden the discovery of genes and their regulatory
behavior, auditory and vestibular studies have begun to focus on proteins in terms of
their interactions, structure, and how these factors relate to function.
In light of the dramatic changes in the auditory and vestibular sciences over these
past fifteen plus years, this book describes RNA, protein, and imaging protocols that
currently are in use and that have provided insights into genetic regulation, as well as
insights into genes and pathogens involved in diseases of the ear. This overview pro-
vides a perspective of basic research with both mammalian and non-mammalian ani-
mal models, as well as protocols applicable to clinical studies. The chapters in Part 1
include basic protocols of RNA isolation and expression, followed by methods to
study cell lineage, gene delivery, and the identificationand use of stem cells. This sec-
tion ends with techniques that are applicable to clinical studies of genes, pathogens,
and cancers that lead to hearing loss in humans. Part 2 focuses on the study of inner
ear proteins and more specifically on their interactions, including techniques such as
the yeast-two hybrid assay, coimmunoprecipitation, plasmon resonance, and protein
taggingformassspectrometry.Thefinalsection,Part3,describesimagingtechniques
v
vi Preface
useful for the study of ions, protein-protein interactions, and imaging of proteins at
the atomic level.
While the chapters are written by specialists in the auditory and vestibular fields,
the techniques described herein will be useful to those exploring genes and proteins
in other systems as well, especially where tissues are scarce and where a comparative
approach lends itself to discovering the underlying causes of human disorders.
Contents
Preface.......................................................................................... v
Contributors..................................................................................... ix
PART I. NUCLEIC ACID PROTOCOLS
1 RNAIsolationfromXenopusInnerEarSensoryEndorgans
for TranscriptionalProfilingandMolecularCloning
CasildaTrujillo-Provencio,TuShunR.Powers,DavidR.Sultemeier,
andElbaE.Serrano.................................................................. 3
2 SynthesisofBiotin-LabeledRNAforGeneExpressionMeasurements
UsingOligonucleotideArrays
AnaE.Va´zquez,LipingNie,andEbenezerN.Yamoah............................... 21
3 InsituHybridizationApproachtoStudymRNAExpression
and DistributioninCochlearFrozenSections
HakimHiel.............................................................................. 31
4 LineageAnalysisofInnerEarCellsUsingGenomicTagsforClonal
Identification
TakunoriSatohandDonnaM.Fekete.................................................. 47
5 GeneticFate-MappingApproaches:NewMeanstoExploretheEmbryonic
OriginsoftheCochlearNucleus
JunChulKimandSusanM.Dymecki.................................................. 65
6 ThePracticalUseofCreandloxPTechnologiesinMouseAuditory
Research
YilingYuandJianZuo................................................................. 87
7 Helios(cid:2)R GeneGun–MediatedTransfectionoftheInnerEarSensory
Epithelium
InnaA.Belyantseva.....................................................................103
8 Electroporation-MediatedGeneTransfertotheDevelopingMouse
Inner Ear
JohnV.Brigande,SamuelP.Gubbels,DavidW.Woessner,
JonathanJ.Jungwirth,andCatherineS.Bresee....................................125
9 IsolationofSphere-FormingStemCellsfromtheMouseInnerEar
KazuoOshima,PascalSenn,andStefanHeller........................................141
10 MolecularBiologyofVestibularSchwannomas
Long-ShengChangandD.BradleyWelling............................................163
11 MultilocusSequenceTypingandPulsedFieldGelElectrophoresisofOtitis
MediaCausingPathogens
JonathanC.ThomasandMelindaM.Pettigrew.......................................179
12 Fluorescence“InSitu”HybridizationfortheDetectionofBiofilm
in the MiddleEarandUpperRespiratoryTractMucosa
LauraNistico,ArminGieseke,PaulStoodley,LuanneHall-Stoodley,
JosephE.Kerschner,andGarthD.Ehrlich...........................................191
vii
viii Contents
13 PositionalCloningofDeafnessGenes
HannieKremerandFransP.M.Cremers..............................................215
PART II. AMINO ACID PROTOCOLS
14 Twist-OffPurificationofHairBundles
Jung-BumShin,JamesPagana,andPeterG.Gillespie................................241
15 YeastTwo-HybridScreeningtoTestforProtein–ProteinInteractions
in theAuditorySystem
DhasakumarS.Navaratnam...........................................................257
16 TheUseof2-DGelstoIdentifyNovelProtein–ProteinInteractions
in the Cochlea
ThandavarayanKathiresan,MargaretC.Harvey,andBerndH.A.Sokolowski.....269
17 IdentificationofFunctionallyImportantResidues/DomainsinMembrane
ProteinsUsinganEvolutionaryApproachCoupledwithSystematic
MutationalAnalysis
LavanyaRajagopalan,FredA.Pereira,OlivierLichtarge,
andWilliamE.Brownell.............................................................287
18 InVivoVerificationofProteinInteractionsintheInnerEar
by Coimmunoprecipitation
MargaretC.HarveyandBerndH.A.Sokolowski......................................299
19 IdentificationofTranscriptionFactor–DNAInteractionsUsingChromatin
ImmunoprecipitationAssays
LipingNie,AnaE.Va´zquez,andEbenezerN.Yamoah...............................311
20 SurfacePlasmonResonance(SPR)AnalysisofBindingInteractions
of ProteinsinInner-EarSensoryEpithelia
DennisG.Drescher,NeeliyathA.Ramakrishnan,MarianJ.Drescher...............323
21 MultiplexedIsobaricTaggingProtocolsforQuantitativeMass
SpectrometryApproachestoAuditoryResearch
DouglasE.Vetter,JohnveslyBasappa,andSevinTurcan..............................345
PART III. IMAGING PROTOCOLS
22 FluorescenceMicroscopyMethodsintheStudyofProteinStructure
and Function
HeatherJensen-Smith,BenjaminCurrall,DanielleRossino,LeAnnTiede,
MichaelNichols,andRichardHallworth............................................369
23 IonImagingintheCochlearHairCells
GregoryI.Frolenkov.....................................................................381
24 AtomicForceMicroscopyinStudiesoftheCochlea
MichioMurakoshiandHiroshiWada...................................................401
Index............................................................................................415
Contributors
JOHNVESLY BASAPPA • DepartmentofNeuroscience,TuftsUniversitySchool
of Medicine,Boston,MA,USA;JeanMayerUSDAHumanNutritionResearch
CenteronAging,TuftsUniversity,Boston,MA, USA
INNA A. BELYANTSEVA • LaboratoryofMolecularGenetics,SectiononHuman
Genetics,NIDCD,NationalInstitutesofHealth,Rockville,MD, USA
CATHERINE S. BRESEE • OregonHearingResearchCenter,OregonHealth&Science
University,Portland,OR, USA
JOHN V. BRIGANDE • OregonHearingResearchCenter,OregonHealth&Science
University,Portland,OR, USA
WILLIAM E. BROWNELL • BobbyR.AlfordDepartmentofOtolaryngology –HNS,
BaylorCollegeofMedicine,Houston,TX, USA
LONG-SHENG CHANG • DepartmentsofPediatrics,Otolaryngology,andPathology,
TheOhioStateUniversityCollegeofMedicine,CenterforChildhoodCancer
ResearchInstituteatNationalChildren’sHospital,Columbus,OH, USA
FRANS P. M. CREMERS • DepartmentofHumanGenetics,NijmegenCentrefor
MolecularLifeSciences,RadboudUniversityNijmegenMedicalCentre,Nijmegen,
TheNetherlands
BENJAMIN CURRALL • DepartmentofBiomedicalSciences,CreightonUniversity
SchoolofMedicine,Omaha,NE, USA
DENNIS G. DRESCHER • DepartmentsofOtolaryngologyandBiochemistry,Wayne
StateUniversitySchoolofMedicine,Detroit,MI, USA
MARIAN J. DRESCHER • DepartmentofOtolaryngology,WayneStateUniversity
SchoolofMedicine,Detroit,MI, USA
SUSAN M. DYMECKI • DepartmentofGenetics,HarvardMedicalSchool,Boston,
MA, USA
GARTH D. EHRLICH • CenterforGenomicSciences,AlleghenySingerResearch
Institute,AlleghenyGeneralHospital,Pittsburgh,PA,USA;Departmentof
MicrobiologyandImmunology,DrexelUniversityCollegeofMedicine,Allegheny
Campus,Pittsburgh,PA, USA
DONNA M. FEKETE • DepartmentofBiologicalSciences,PurdueUniversity,West
Lafayette,IN, USA
GREGORY I. FROLENKOV • DepartmentofPhysiology,UniversityofKentucky,
ChandlerMedicalCenter,Lexington,KY, USA
ARMIN GIESEKE • Max-PlanckInstituteforMarineMicrobiology,Bremen,Germany
PETER G. GILLESPIE • OregonHearingResearchCenterandVollumInstitute,
OregonHealth&ScienceUniversity,Portland,OR, USA
SAMUEL P. GUBBELS • UniversityofIowaHospitals,IowaCity,IA, USA
LUANNE HALL-STOODLEY • CenterforGenomicSciences,AlleghenySingerResearch
Institute,AlleghenyGeneralHospital,Pittsburgh,PA,USA;Departmentof
ix
x Contributors
MicrobiologyandImmunology,DrexelUniversityCollegeof Medicine,Allegheny
Campus,Pittsburgh,PA, USA
RICHARD HALLWORTH • DepartmentofBiomedicalSciences,CreightonUniversity
Schoolof Medicine,Omaha,NE, USA
MARGARET C. HARVEY • DepartmentofOtolaryngology –HNS,University
of SouthFlorida,CollegeofMedicine,Tampa,FL, USA
STEFAN HELLER • DepartmentsofOtolaryngology–HNSandMolecular&Cellular
Physiology,StanfordUniversitySchoolofMedicine,Stanford,CA, USA
HAKIM HIEL • DepartmentofOtolaryngology –HNS,JohnsHopkinsUniversity,
SchoolofMedicine,Baltimore,MD, USA
HEATHER JENSEN-SMITH • DepartmentofBiomedicalSciences,Creighton
UniversitySchoolofMedicine,Omaha,NE, USA
JONATHAN J. JUNGWIRTH • OregonHearingResearchCenter,OregonHealth&
ScienceUniversity,Portland,OR, USA
THANDAVARAYAN KATHIRESAN •DepartmentofOtolaryngology –HNS,University
ofSouthFlorida,CollegeofMedicine,Tampa,FL, USA
JOSEPH E. KERSCHNER • DepartmentofOtolaryngology,MedicalCollegeof
Wisconsin,Milwaukee,WI, USA
JUN CHUL KIM • DepartmentofGenetics,HarvardMedicalSchool,Boston,
MA, USA
HANNIE KREMER • DepartmentofOtorhinolaryngology,NijmegenCentrefor
MolecularLifeSciences,RadboudUniversityNijmegenMedicalCentre,Nijmegen,
TheNetherlands
OLIVIER LICHTARGE • DepartmentofMolecularandHumanGenetics,Baylor
CollegeofMedicine,Houston,TX, USA
MICHIO MURAKOSHI • DepartmentofBioengineeringandRobotics,Graduate
SchoolofEngineering,TohokuUniversity,Sendai, Japan
DHASAKUMAR S. NAVARATNAM • DepartmentofNeurology,YaleSchoolof
Medicine,NewHaven,CT, USA
MICHAEL NICHOLS • DepartmentofPhysics,CreightonUniversity,Omaha,
NE, USA
LIPING NIE • DepartmentofOtolaryngology,CenterforNeuroscience,Universityof
California,Davis,CA, USA
LAURA NISTICO • CenterforGenomicSciences,Allegheny-SingerResearchInstitute,
AlleghenyGeneralHospital,Pittsburgh,PA, USA
KAZUO OSHIMA • DepartmentsofOtolaryngology–HNSandMolecular&Cellular
Physiology,StanfordUniversitySchoolofMedicine,Stanford,CA, USA
JAMES PAGANA • OregonHearingResearchCenterandVollumInstitute,Oregon
Health&ScienceUniversity,Portland,OR, USA
FRED A. PEREIRA • BobbyR.AlfordDepartmentofOtolaryngology –HNS,
HuffingtonCenteronAgingandDepartmentofMolecularandCellularBiology,
BaylorCollegeofMedicine,Houston,TX, USA
MELINDA M. PETTIGREW • DivisionofEpidemiologyofMicrobialDiseases,Yale
UniversitySchoolofPublicHealth,NewHaven,CT, USA