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  • ForwardGeneticScreens:Strategiesandchallenges

    HarwinGoFish

    22July2015

  • Forwardgenetics

    Phenotype Gene

    Severaladvantages: Startingpointisastrongphenotype

    Unbiasedapproach possibilitytofindnewregulatorsofcertainprocess

    Abletoobtainlargenumberofgenesinvolvedinthesameprocess

  • Thingstoconsider

    1. Phenotypestoscreenfor2. Methodsofmutagenesis3. Identificationofmutagenizedgene4. Degreeofsaturation5. Proofofcandidategene

  • 1.Phenotypestoscreenfor

    1. Morphology,lethality2. Maternalphenotypes3. Specificembryonicphenotype(Ab staining,

    ISH,nervousandhematopoieticsystems)4. Modifierscreens5. Temperaturesensitivescreen6. Regulatoryelements

  • 2.Methodsofmutagenesis

    Gammarayirradiation

    Chemicalmutagenesis

    Insertional mutagenesis

  • Gammarayirradiation

    Kimmel,1989

  • Time:Mutagenizedspermorembryosatmidblastulastage

    Effect:Largedeletions/translocations

    Pros:RelativelyfastHighmutagenicrate(~1.2hitper100,000genome/rad,higherwithincreasingrad)

    Cons:Highlethality(nonspecific)Hardtomap/maintainlines

    Gammarayirradiation

  • Chemicalmutagenesis

    LawsonandWolfe,2011

  • ChemicalmutagenesisTime:Premeiotic sperm

    Effect:Pointmutations

    Pros:FastmutagenesisandfamilygenerationHighestmutagenicrate(3hits/gene/1000genomesscreened)Morerandomthaninsertional mutagenesis

    Cons:LotsofsilentmissensemutationsPositionalcloningtakesFOREVERNeedmultipleoutcrossestodivergentbackgroundformapping

  • Insertional mutagenesis

    LawsonandWolfe,2011

  • Insertional mutagenesis

    Amsterdametal.,1999

  • Insertional mutagenesisTime:Midblastula embryos

    Effect:Largeinsertionandgenesilencing

    Pros:PositionalcloningissupereasyEveryintegrationresultsinsilencing

    Cons:MutagenesisrateislowerthanENUMutagenesisisverylaborintensiveSlightbiastowardsopenregionsofthegenome(higherinsertionrateat5ends)

  • Summary:mutagenesismethods

    AmsterdamandHopkins,2006

  • 3.Identificationofmutagenizedgene

    Insertional mutagenesishastheupperhand!

    InversePCR+BLASTing knownsequence=rapidmapping!

    Sometechnicalproblemswithhighlysimilarregionsinthepast,butwithbettergenomesequencethisisminimized

    AmsterdamandHopkins,2006

  • PositionalCloning

    3.Identificationofmutagenizedgene

    Chromosomeswithmutation

    mutation

    Polymorphicmarkersclosetomutationwillsegregatewithmutationmorefrequentlythanmarkersfurtheraway

    =SimpleSequenceLengthPolymorphisms(SSLPs)

  • Positionalcloning

    ZhouandZon,2011

  • Laborintensive Need~2000mutantstobeabletomapto0.1cM

    Withbettergenome,stillneeds~400mutantstomapto1cM,andsequencegenesinbetween

    Therearesiteswithminimalrecombinationinthegenome!

    Positionalcloning

  • Mutagenesis+Wholegenomeseq

    Zebrafish MutationProject(ZMP)

  • ProblemswithZMP

    1. Lowdepthofsequence Sequencinggametesislesssensitive

    2. Difficultyinrecoveringfoundmutations,husbandry

    3. Spaceconstraints

  • Positionalcloning+Seq:Abetterapproach?

    Obholzer etal.,2012

  • 4.DegreeofSaturationA.Howefficientisthemutagenesis?

    Mullinsetal.,1994B.Howmanyhitsonthesamegene?

    Alsodependsonthegenesize

  • 5.ProofofCandidateGenes

    1. Incaseofmultiplealleles:complementation2. Rescueassays3. Morpholino4. Reversegenetics

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