4/3/2014

An Introduction to Reverse Genetic Tools for Investigating Gene Function

K12 Introductory Advanced IllustratedGlossary InstructorCommunication Translations ThisSection:

GoTo... APS>Education>Advanced>TopicsinPlantPathology> Share AnIntroductiontoReverseGeneticToolsfor InvestigatingGeneFunction

AnIntroductiontoReverseGenetic Tools forInvestigatingGeneFunction

Tierney,M.B.andLamour,K.H.2005.AnIntroductiontoReverseGeneticToolsfor InvestigatingGeneFunction.ThePlantHealthInstructor.DOI:10.1094/PHIA20051025 01.

MelindaB.Tierney1andKurtH.Lamour2
1UniversityofTennesseeandOakRidgeNational

LaboratoryGenomeScienceandTechnologyGraduate ProgramKnoxville,TN
2TheUniversityofTennessee,DepartmentofEntomology

andPlantPathologyKnoxville,TN

Introduction
Geneticsbeganinthe1860swithGregorMendel,an Augustinianmonkwhoperformedexperimentsthat suggestedtheexistenceofgenes(Griffithsetal.2000).The discoveryofthestructureofDNAbyJamesWatsonand FrancisCrickinthe1950s(WatsonandCrick1953), followedbythedevelopmentofdideoxyterminator sequencingbySangerinthelate1970s(Sanger,Nicklen, andCoulson1977)andthenofPCR(polymerasechain reaction)technologybyKaryMullisinthe1980s(Mullisand Faloona1987Saikietal.1985)setoffageneticrevolution. Technologicaladvancesinsequencinghavegreatly acceleratedouraccumulationofgeneticsequencedatato thepointnowwherewholegenomesequencesarepublicly availableforalargenumberoforganismsincludingplant pathogens.Thetoolboxforgeneticresearchisexpanding rapidly,andthisoverviewpresentsasuiteoftoolsgenerally referredtoasreversegeneticsthatcanbeusedto investigategenefunction.

ForwardGeneticsvs.ReverseGenetics
Variantshelpusunderstandthe'normal.'Variationcanbe measuredatmanyscalesfrommacro(bodysize, morphology)todifferentlevelsofmicrovariation(crude
https://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx 1/10

4/3/2014

An Introduction to Reverse Genetic Tools for Investigating Gene Function

proteinprofilestoDNAsequencevariation).Forward geneticsreferstoaprocesswherestudiesareinitiatedto determinethegeneticunderpinningsofobservable phenotypicvariation.Inmanycasestheobservablevariation hasbeeninducedusingaDNAdamagingagent(mutagen) butalsomaybenaturallyoccurring.Theinvestigator eventuallyendsupsequencingthegeneorgenesthoughtto beinvolved(Figure1).

Figure1 Withtheadventofwholegenomesequencingmany researchersarenowinaverydifferentposition.Theyhave accesstoallofthegenesequenceswithinagivenorganism andwouldliketoknowtheirfunction.So,insteadofgoing fromphenotypetosequenceasinforwardgenetics,reverse geneticsworksintheoppositedirectionagenesequence isknown,butitsexactfunctionisuncertain.Inreverse genetics,aspecificgeneorgeneproductisdisruptedor modifiedandthenthephenotypeismeasured(Figure1). Herewewilloverviewsomeofthetechniquesforreverse geneticswithaspecialemphasisontheTILLING(Targeting InducedLocalLesionsINGenomes)techniquewhichis beingutilizedonplantpathogensinthegenusPhytophthora.

SpecificReverseGeneticapproaches
Thegoalinreversegeneticsistoinvestigatetheimpactof inducedvariationwithinaspecificgeneandtoinfergene function.Theprocessofdisruptionoralterationcaneitherbe targetedspecificallyasinthecaseofgenesilencingor homologousrecombinationorcanrelyonnontargeted randomdisruptions(e.g.,chemicalmutagenesis,transposon mediatedmutagenesis)followedbyscreeningalibraryof individualsforlesionsataspecificlocation.Followingisa briefoverviewofsomecommonlyusedtargetedandnon targetedapproaches.

GeneSilencing
RNAinterference(RNAi)istheprocessbywhichexpression ofatargetgeneisinhibitedbyantisenseandsenseRNAs.It worksbasedontheabilityofdoublestrandedsequencesto
https://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx 2/10

4/3/2014

An Introduction to Reverse Genetic Tools for Investigating Gene Function

recognizeanddegradesequencesthatarecomplementary tothem(Lewin2004).RNAiwasfirstdiscoveredin Caenorhabditiseleganswhentheintroductionofdouble strandedRNAwasobservedtobeanefficientmethodfor silencinggeneexpression(Fireetal.1998Kuttenkeulerand Boutros2004).RNAibasedsilencingisanexcitingstrategy forreversegenetics(Waterhouse,Graham,andWang1998). RNAinterferencehasrecentlybecomeapowerfultoolto silencetheexpressionofgenesandanalyzetheirlossof functionphenotype,allowinganalysisofgenefunctionwhen mutantallelesarenotavailable.Havingbeenshowntowork inasimilarmannerinallmetazoans,RNAihasproventobe applicabletomanyorganismsandhasbeenusedto generateawidevarietyoflossoffunctionphenotypes (KuttenkeulerandBoutros2004).Thephenomenonofpost transcriptionalgenesilencingobservedinplantsmayalso beduetoarelatedRNAimechanism(Waterhouse,Graham, andWang1998).RNAibasedsilencingutilizesthe endonucleaseDicertocleavesinglestrandedRNAs, abbreviatedsiRNAs,fromdoublestrandedRNAtheRISC complexthendestroysspecifictargetmRNAsbasedon sequencecomplementaritywiththesiRNA(Pattanayaketal. 2005). RNAihasbeenusedforasystematicanalysisofgene functioninC.elegansbygeneratinglossoffunction phenotypes,creatingalibraryofwormsexpressingdsDNA correspondingtodifferentgenes(Lewin2004).Genome wideRNAiscreensagainsttheselibrariesofpredicted geneshaveallowedstudyofavarietyofbiological processesinC.elegans.Agenomewidelibraryofdouble strandedRNAsthattargeteverygeneintheDrosophila genomehasalsobeenpublishedthatissuitableforhigh throughputcellbasedassays(KuttenkeulerandBoutros 2004).OnedifficultyinusingRNAiasareversegenetic techniqueisthatthroughputislimitedbytheabilitytodeliver siRNAstotargetloci(Henikoff,Till,andComai2004).Itis alsolaborintensive,cangiveambiguousresults,andcanbe unsuitableforisolatingmutantsthathavelethalorsterile phenotypes(GilchristandHaughn2005).

Targetedgenedisruptionbyhomologous recombination
HomologousrecombinationisareciprocalexchangeofDNA sequences,asinbetweentwochromosomesthatcarrythe samegeneticloci(Lewin2004).Justashomologous recombinationhasbeenfoundtobemainlyinitiatedwitha doublestrandbreak,genetargetingbyhomologous recombinationisassociatedwiththerepairofdoublestrand breaks.Thedoublestrandbreakrepairandsynthesis dependentstrandannealingmodelsarethemostgenerally acceptedmodelstoexplaingenetargeting(IidaandTerada 2004). Homologousrecombinationhasbeenwidelyusedin embryonicstem(ES)cellsinmice,andhasallowed constructionofprecisemutationsinnearlyeverygene.A

https://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx

3/10

4/3/2014

An Introduction to Reverse Genetic Tools for Investigating Gene Function

constructionofprecisemutationsinnearlyeverygene.A reversegeneticsystemusinghomologousrecombination hasrecentlybeendevelopedforDrosophila.Itispromising, butisalengthyprocedureandrequiresgenerationof specifictransgenicflies(Stemple2004).Reproduciblegene targetingbyhomologousrecombinationisnowalsofeasible inrice.Withthecombinationofsitespecificrecombination systems(suchasCrelox),thefutureofgenetargetingby homologousrecombinationasaroutineprocedurefor engineeringthegenomeofriceandpresumablyotherplants isbright(IidaandTerada2004).

Insertionalmutagenesis/transposonmediated mutagenesis
Transposonsaremobilegeneticelementsthatcanrelocate fromonegenomiclocationtoanother(Hayes2003).They areDNAsequencesthatcaninsertthemselvesatanew locationinthegenomewithouthavinganysequence relationshipwiththetargetlocus(Lewin2004).Transposon basedsignaturetaggedmutagenesishasbeensuccessfulin identifyingessentialgenesaswellasgenesinvolvedin infectivityofavarietyofpathogens.Strategiesforinsertional mutagenesisusingtransposonshavebeendevelopedfora numberofanimalandplantmodels(Hayes2003).Reverse geneticsiscurrentlybeingdoneinDrosophilaandC. elegansbyutilizinglibrariesofindividualswhocarry transposableelementinsertions,manyofwhichhavebeen mapped,andsomeofwhichwilldisrupttheexpressionof nearbygenes.InDrosophilaPelements,impreciseexcision canbedriventogenerateamutationinthenearestgene. Transposonbasedmethodsarealsobeingusedin Arabidopsis,maizeandotherplants(Stemple2004).One drawbackofinsertionalmutagenesisisthelowfrequencyof mutations,necessitatingthescreeningoflargenumbersof individualstofindmutationsinanygivengene(Gilchristand Haughn2005).Also,insertionsinessentialgeneswill usuallycauselethality,andlessseveremutationsmustbe generatedinthesegenesinordertounderstandgene function(Tilletal.2003). ThesegmentoftheTiplasmidofAgrobacteriumtumefaciens knownasTDNAthatcarriesgenestotransformtheplant cellhasalsobeenutilizedforinsertionalmutagenesis.T DNAinsertionalmutagenesishasbeenusedtoobtaingene knockoutsforgreaterthan70%ofArabidopsisgenes (Alonsoetal2003),butnocomparableresourcesexistfor riceormaizeevenashighcoveragegenomicsequenceis becomingincreasinglyavailable(Henikoff,Till,andComai 2004).Unlikeothersuccessfulgenetargetingsystems (namelymouse,Physcomitrella,andDrosophila),theprecise mechanismofTDNAintegrationintotheplantgenome remainslargelyunknown(IidaandTerada2004).LikeRNA suppressiontechniques,insertionalmutagenesisislimited byitshostrangeandbyitslimitedrangeofalleletypes (McCallumetal.2000). Genereplacementviatransformationisacommonlyused toolformanyfilamentousfungi(Fang,Hanau,and
https://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx 4/10

4/3/2014

An Introduction to Reverse Genetic Tools for Investigating Gene Function

Vaillancourt2002LalucqueandSilar2004Takanoetal. 2000).Thetransformationcanbemediatedinmanyways, includingAgrobacterium(Zhangetal.2003)andvarious othertransformationvectors(ScottCraigetal.1998Takano etal.2000).

Chemicalmutagenesis/TILLING
Twoofthemostwidelyusedmutagensforchemical mutagenesisexperimentsareEMS(ethylmethanesulfonate) andENU(ethylnitrosourea).EMSisachemicalmutagenthat alkylatesguaninebases.Thealkylatedguaninewillthen pairwiththymineinsteadofthepreferredcytosinebase, ultimatelyresultinginaG/CtoA/Ttransition.EMSisthe mostcommonlyusedmutangeninplants.InArabidopsis, fivepercentofEMSinducedmutationsintargetedcoding regionsresultinprematureterminationofthegeneproduct, whilefiftypercentresultinmissensemutationsthatalterthe aminoacidsequenceoftheencodedprotein(Gilchristand Haughn2005).Thishighlevelofmissensemutations relativetoterminatedgeneproductsisveryusefulin analyzinggenefunction.ENUalsoinducespointmutations, andisamorepotentmutagenthanEMS.Itisalsoan alkylatingagent,mutagenizingbytransferringanethylgroup tooxygenornitrogenradicalsintheDNAmolecule,which leadstomispairingandultimatelyresultsinbasepair substitutions,andsometimesbasepairlossesifnotrepaired (Gunet2004). Chemicalmutagenesisisattractiveforreversegenetics becauseitresultsininducedpointmutations,whichcreatea diverserangeofallelesforgeneticanalysis.Itinducesa largenumberofrecessivemutationspergenomethatare randomlydistributed(GilchristandHaughn2005).Because chemicalmutagenesisisalreadywidelyusedinmany organismsforforwardgeneticscreens,itpromisestobe generallyapplicableforreversegenetics(Henikoff,Till,and Comai2004).Untilrecently,chemicalmutagenesishasnot beenwidelyusedasatoolforreversegeneticsbecauseof thelackofhighthroughputtechniquesfordetectingpoint mutations(GilchristandHaughn2005).TheTILLING strategyprovidesahighthroughputstrategytodetectsingle basechangeswithingenetictargetsandcanbeappliedtoa widevarietyoforganisms.

TILLINGPhytophthora
Wholegenomesequencesforthesoybeanpathogen PhytophthorasojaeandthesuddenoakdeathpathogenP. ramorumweremadepublicneartheendof2004.Large scalegenomicsequencingprojectsareunderwayforthe potatolateblightpathogenP.infestansandthevegetable pathogenP.capsici.Thecompilationofthesegenomic sequencesallowstheapplicationofmanysophisticated researchtoolsandpromisesabetterunderstandingofthis devastatinggroupofpathogens.Inanefforttobetter understandgenefunctionwithinPhytophthoraaprojectto developareversegeneticTILLINGresourcefor Phytophthorahasbeeninitiated.Figure2providesan
https://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx 5/10

4/3/2014

An Introduction to Reverse Genetic Tools for Investigating Gene Function

overviewoftheprocessasitisbeingappliedto Phytophthora.Zoosporespresentanideallifestagefor mutagenesisastheyareuninucleateandsinglemutant individualscanreadilybeisolatedfollowingmutagenesis. ThemutationrateforEMSorENUisnotknownfor Phytophthoraandlethalityisbeingusedasanindicatorof doseresponse.GenomicDNAisextractedfromthemutant individualsandpooled2to4fold.ThegenomicDNAlibrary canthenberepeatedlyscreened.Specificgenesare amplifiedfromthepoolsofgenomicDNA,andthePCR productsareheatedupandallowedtocoolslowlytoform heteroduplexesbetweenwildtypeandmutantstrandsof DNA.Theheteroduplexesaretreatedwiththesinglestrand specificendonucleaseCEL1whichcuts3'ofsinglebase mismatchesproducingnovelfragmentsofDNA.CEL1 treatedPCRproductsarethenresolvedonapolyacrylamide gelandscreenedforthepresenceofnovelfragments.Pools containingnovelfragmentsarethenanalyzedtodetermine exactlywhichmutantiscarryinganinducedpointmutation, andthePCRproductfromthismutantissequencedto determineiftheinducedchangeispredictedtobesilent, missense,oraknockoutmutation.

https://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx

6/10

4/3/2014

An Introduction to Reverse Genetic Tools for Investigating Gene Function

Figure2 SincePhytophthoraisdiploidthroughmostofitslifecycle (includingthezoosporestage)alloftheinducedpoint mutationsexistintheheterozygousstate.Onceanonsilent pointmutationhasbeenidentifiedthemutantisolateistaken throughthesexualstageandthesexualprogenyare screenedtoidentifyindividualshomozygousforthemutation underinvestigation.Homozygousmutantsarethentestedto determineifthereisanalteredphenotype.

Limitations/roadblockstocompletingreverse genetics
Completingreversegeneticsisnotwithoutitspitfalls,and notalltechniquescanbeappliedtoallorganisms.Tobe successful,thereareseveralaspectsthatmustbechecked. Fororganismsthatdonothaveefficienttransformation systemsavailabletechniquessuchasTILLINGthatcanbe appliedwithouttransformationmaybetheonlypractical choice.Inthesecases,therateofmutagenesisisan importantfactorthatcanbedifficulttodetermine.Theloadof mutationsmustbebalancedwiththerecoveryofmutants (Tilletal.2003)inotherwords,thegenomecan'tbeso riddledwithmutationsthatitisimpossibletoseeamutant phenotype.Alsotobeconsideredisthefertilityofthe mutagenizedorganism,especiallyinthefirstgeneration,but alsoinsubsequentgenerations(Perryetal.2003),both beforeandaftermutagenesis.Thisisespeciallytruefor diploidorganisms,becauseifthesexualmachineryisnot intactandworkingproperly,thenitisimpossibletoobtaina homozygousmutant.Themutagenizedorganismsmustalso bekeptalivelongenoughtoscreenamutantpopulationfor aspecifictarget.Forsomeorganisms,likeArabidopsisor Phytophthora,thisisnotaproblemastheseedsorcultures arerelativelyeasytostore.Itpresentsachallengeforother organisms,suchaszebrafishorrats,becausetheymustbe storedandkeptalivethroughthemutantscreeningstage.

Supplementaryinformation:examplesof TILLINGinAction
*Arabidopsis
ApublicTILLINGresourcewassetupin2001forthe Arabidopsiscommunity.Thiseffortwascalledthe ArabidopsisTILLINGProject(ATP)andisnowcalledthe SeattleTillingProject(STP)andisajointeffortbetweenthe ComaiLaboratoryattheUniversityofWashingtonandthe HenikoffLaboratoryattheFredHutchinsonCancer
https://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx 7/10

4/3/2014

An Introduction to Reverse Genetic Tools for Investigating Gene Function

ResearchCenterinSeattle,Washington.Usersarecharged afeethatcoverspartialcostsoftheservicesprovidedto requestmutationsingenesofinterest(GilchristandHaughn 2005Tilletal.2003).Trainingsessions,workshops,andon goingsupporttoresearchersinterestedindeveloping TILLINGinotherorganismshasbeenmadeavailable throughtheATPandhasservedtomaketheTILLING techniquemorewidespread.Thisgrouphasalsodeveloped andmadepubliclyavailablewebbasedsoftwareprograms forPCRprimerdesignandvisualizationofpolymorphisms (GilchristandHaughn2005).

*Zebrafish
TargetselectedmutagenesisusingTILLINGisalsobeing doneinzebrafish.TheHubrechtLaboratoryatThe NetherlandsInstituteforDevelopmentalBiologyhas generatedalibraryof4608ENUmutagenizedF1animals andhaskeptalivingstock.TheDNAfromtheseanimalshas beenscreenedformutationsin16genesusingTILLING followedbyresequencing.Thisresultedin255mutations beingidentified,14ofwhichresultedinaprematurestop codon,7inasplicedonor/acceptorsitemutation,and119in anaminoacidchange.Theywereabletoknockout13 differentgenesinonlyafewmonthstimethroughreverse genetics(Wienholdsetal.2003).Thusfar,mutant phenotypeshavebeencharacterizedfortwoofthezebrafish genesmodifiedviaTILLINGthegeneforDicer1(Stemple 2004Wienholdsetal.2003)andthegeneforadenomatous polyposis,atumorsuppressor,whichthroughwasfoundto haveapreviouslyunknownfunctionforsignalingincardiac valveformation(Hurlstoneetal.2003Stemple2004).

Acknowledgements
ThisworkwassupportedbyaNationalScienceFoundation, FacultyEarlyCareer(CAREER)Developmentaward (#0347624)toKurtLamour.

LiteratureCited
1. Alonso,J.M.,Stepanova,A.N.,Leisse,T.J.,Kim,C.J., Chen,H.,Shinn,P.,Stevenson,D.K.,Zimmerman,J., Barajas,P.,Cheuk,R.,Gadrinab,C.,Heller,C.,Jeske, A.,Koesema,E.,Meyers,C.C.,Parker,H.,Prednis,L., Ansari,Y.,Choy,N.,Deen,H.,Geralt,M.,Hazari,N., Hom,E.,Karnes,M.,Mulholland,C.,Ndubaku,R., Schmidt,I.,Guzman,P.,AguilarHenonin,L.,Schmid, M.,Weigel,D.,Carter,D.E.,Marchand,T.,Risseeuw, E.,Brodgden,D.,Zeko,A.,Crosby,W.L.,Berry,C.C., andEcker,J.R.2003.GenomeWideInsertional MutagenesisofArabidopsisthaliana.Science 301:653657. 2. Fang,G.C.,Hanau,R.M.,andVaillancourt,L.J.2002. TheSOD2gene,encodingamanganesetype superoxidedismutase,isupregulatedduring conidiogenesisintheplantpathogenicfungus
https://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx 8/10

4/3/2014

An Introduction to Reverse Genetic Tools for Investigating Gene Function

conidiogenesisintheplantpathogenicfungus Colletotrichumgraminicola.FungalGeneticsand Biology36:155165. 3. Fire,A.,XU,Siqun,Montgomery,M.K.,Kostas,S.A., Driver,S.E.,andMello,C.C.1998.Potentandspecific geneticinterferencebydoublestrandedRNAin Caenorhabditiselegans.Nature391:806811. 4. Gilchrist,E.J.,andHaughn,G.W.2005.TILLING withoutaplough:anewmethodwithapplicationsfor reversegenetics.CurrentOpinioninPlantBiology 8:211215. 5. Griffiths,A.J.F.,Miller,J.H.,Suzuki,D.T.,Lewontin, R.C.,andGelbart,W.M.2000.AnIntroductionto GeneticAnalysis.7ed.NewYork:W.H.Freemanand Company. 6. Gunet,J.2004.ChemicalMutagenesisofthemouse genome:anoverview.Genetica122:924. 7. Hayes,F.2003.TransposonBasedStrategiesfor MicrobialFunctionalGenomicsandProteomics. AnnualReviewofGenetics37:329. 8. Henikoff,S.,Till,B.J.,andComai,L.2004.TILLING. TraditionalMutagenesisMeetsFunctionalGenomics. PlantPhysiology135:17. 9. Hurlstone,A.F.L.,Haramis,A.G.,Wienholds,E., Begthel,H.,Korving,J.,Eeden,F.van,Cuppen,E., Zivkovic,D.,Plasterk,R.H.A.,andClevers,H.2003. TheWnt/Betacateninpathwayregulatescardiacvalve formation.Nature425:633637. 10. Iida,S.,andTerada,R.2004.Ataleoftwo integrations,transgeneandTDNA:genetargetingby homologousrecombinationinrice.Currentopinionin Biotechnology15:132138. 11. Kuttenkeuler,D.,andBoutros,M.2004.Genomewide RNAiasaroutetogenefunctioninDrosophila. BriefingsinFunctionalGenomicsandProteomics3 (2):168176. 12. Lalucque,H.,andSilar,P.2004.Incomplete PenetranceandVariableExpressivityofaGrowth DefectasaConsequenceofKnockingOutTwoK+ TransportersintheEuascomyceteFungusPodospora anserina.Genetics166:125133. 13. Lewin,B.2004.GenesVII.UpperSaddleRiver,NJ: PearsonPrenticeHall. 14. McCallum,C.M.,Comai,L.,Greene,E.A.,andHenikoff, S.2000.TargetingInducedLocalLesionsINGenomes (TILLING)forPlantFunctionalGenomics.Plant Physiology123:439442. 15. Mullis,K.B.,andFaloona,F.A.1987.Specific synthesisofDNAinvitroviaapolymerasecatalyzed chainreaction.MethodsinEnzymology155:335350. 16. Pattanayak,D.,Agarwal,S.,Sumathi,S.,Chakrabarti, S.K.,Naik,P.S.,andKhurana,S.M.2005.Smallbut mightyRNAmediatedinterferenceinplants.Indian JournalofExperimentalBiology43(1):724. 17. Perry,J.A.,Wang,T.L.,Welham,T.J.,Gardner,S., Pike,J.M.,Yoshida,S.,andParniske,M.2003.A TILLILNGReverseGeneticsToolandaWeb AccessibleCollectionofMutantsoftheLegumeLotus japonicus.PlantPhysiology131:866871. 18. Saiki,R.K.,Scharf,S.,Faloona,F.,Mullis,K.B.,Horn, G.T.,Erlich,H.A.,andArnheim,N.1985.Enzymatic AmplificationofBetaGlobinGenomicSequencesand RestrictionSiteAnalysisforDiagnosisofSickleCell Anemia.Science230:13501354. 19. Sanger,F.,Nicklen,S.,andCoulson,A.R.1977.DNA Sequencingwithchainterminatinginhibitors. ProceedingsoftheNationalAcademyofScience74

https://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx

9/10

4/3/2014

An Introduction to Reverse Genetic Tools for Investigating Gene Function

ProceedingsoftheNationalAcademyofScience74 (12):54635467. 20. ScottCraig,J.S.,Cheng,Y.,Cervone,F.,Lorenzo,G. De,Pitkin,J.W.,andWalton,J.D.1998.Targeted MutantsofCochlioboluscarbonumLackingtheTwo MajorExtracellularPolygalacturonases.Appliedand EnvironmentalMicrobiology:14971503. 21. Stemple,D.L.2004.TILLINGahighthroughput harvestforfunctionalgenomics.NatureReviews Genetics5:145150. 22. Takano,Y.,Kikuchi,T.,Kubo,Y.,Hamer,J.E.,Mise,K., andFurusawa,I.2000.TheColletotrichumlagenarium MAPKinaseGeneCMK1RegulatesDiverseAspects ofFungalPathogenesis.MolecularPlantMicrobe Interactions13(4):374383. 23. Till,B.J.,Reynolds,S.H.,Greene,E.A.,Codomo,C.A., Enns,L.C.,Johnson,J.E.,Burtner,C.,Odden,A.R., Young,K.,Taylor,N.E.,Henikoff,J.G.,Comai,L.,and Henikoff,S.2003.LargeScaleDiscoveryofInduced PointMutationsWithHighThrougputTILLING. GenomeResearch13:524530. 24. Waterhouse,P.M.,Graham,M.W.,andWang,M.1998. Virusresistanceandgenesilencinginplantscanbe inducedbysimultaneousexpressionofsenseand antisenseRNA.ProceedingsoftheNationalAcademy ofScience95:1395913964. 25. Watson,J.D.,andCrick,F.H.C.1953.AStructurefor DeoxyriboseNucleicAcid.Nature171:737738. 26. Wienholds,E.,Eeden,F.van,Kosters,M.,Mudde,J., Plasterk,R.H.A.,andCuppen,E.2003.Efficient TargetSelectedMutagenesisinZebrafish.Genome Research13:27002707. 27. Zhang,A.,Lu,P.,DahlRoshak,A.M.,Paress,P.S., Kennedy,S.,Tkacz,J.S.,andAn,Z.2003.Efficient disruptionofapolyketidesynthasegene(pks1) requiredformelaninsynthesisthroughAgrobacterium mediatedtransformationofGlarealozoyensis. MolecularGeneticsandGenomics268:645655.

2014TheAmericanPhytopathologicalSociety.Allrightsreserved.|ContactUsReportaBadLink

https://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx

10/10