Forward Genetic Screens: Strategies challenges ?· Forward Genetic Screens: Strategies and challenges…

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<ul><li><p>ForwardGeneticScreens:Strategiesandchallenges</p><p>HarwinGoFish</p><p>22July2015</p></li><li><p>Forwardgenetics</p><p>Phenotype Gene</p><p>Severaladvantages: Startingpointisastrongphenotype</p><p> Unbiasedapproach possibilitytofindnewregulatorsofcertainprocess</p><p> Abletoobtainlargenumberofgenesinvolvedinthesameprocess</p></li><li><p>Thingstoconsider</p><p>1. Phenotypestoscreenfor2. Methodsofmutagenesis3. Identificationofmutagenizedgene4. Degreeofsaturation5. Proofofcandidategene</p></li><li><p>1.Phenotypestoscreenfor</p><p>1. Morphology,lethality2. Maternalphenotypes3. Specificembryonicphenotype(Ab staining,</p><p>ISH,nervousandhematopoieticsystems)4. Modifierscreens5. Temperaturesensitivescreen6. Regulatoryelements</p></li><li><p>2.Methodsofmutagenesis</p><p>Gammarayirradiation</p><p>Chemicalmutagenesis</p><p>Insertional mutagenesis</p></li><li><p>Gammarayirradiation</p><p>Kimmel,1989</p></li><li><p>Time:Mutagenizedspermorembryosatmidblastulastage</p><p>Effect:Largedeletions/translocations</p><p>Pros:RelativelyfastHighmutagenicrate(~1.2hitper100,000genome/rad,higherwithincreasingrad)</p><p>Cons:Highlethality(nonspecific)Hardtomap/maintainlines</p><p>Gammarayirradiation</p></li><li><p>Chemicalmutagenesis</p><p>LawsonandWolfe,2011</p></li><li><p>ChemicalmutagenesisTime:Premeiotic sperm</p><p>Effect:Pointmutations</p><p>Pros:FastmutagenesisandfamilygenerationHighestmutagenicrate(3hits/gene/1000genomesscreened)Morerandomthaninsertional mutagenesis</p><p>Cons:LotsofsilentmissensemutationsPositionalcloningtakesFOREVERNeedmultipleoutcrossestodivergentbackgroundformapping</p></li><li><p>Insertional mutagenesis</p><p>LawsonandWolfe,2011</p></li><li><p>Insertional mutagenesis</p><p>Amsterdametal.,1999</p></li><li><p>Insertional mutagenesisTime:Midblastula embryos</p><p>Effect:Largeinsertionandgenesilencing</p><p>Pros:PositionalcloningissupereasyEveryintegrationresultsinsilencing</p><p>Cons:MutagenesisrateislowerthanENUMutagenesisisverylaborintensiveSlightbiastowardsopenregionsofthegenome(higherinsertionrateat5ends)</p></li><li><p>Summary:mutagenesismethods</p><p>AmsterdamandHopkins,2006</p></li><li><p>3.Identificationofmutagenizedgene</p><p>Insertional mutagenesishastheupperhand!</p><p>InversePCR+BLASTing knownsequence=rapidmapping!</p><p>Sometechnicalproblemswithhighlysimilarregionsinthepast,butwithbettergenomesequencethisisminimized</p><p>AmsterdamandHopkins,2006</p></li><li><p>PositionalCloning</p><p>3.Identificationofmutagenizedgene</p><p>Chromosomeswithmutation</p><p>mutation</p><p> Polymorphicmarkersclosetomutationwillsegregatewithmutationmorefrequentlythanmarkersfurtheraway</p><p>=SimpleSequenceLengthPolymorphisms(SSLPs)</p></li><li><p>Positionalcloning</p><p>ZhouandZon,2011</p></li><li><p> Laborintensive Need~2000mutantstobeabletomapto0.1cM</p><p> Withbettergenome,stillneeds~400mutantstomapto1cM,andsequencegenesinbetween</p><p> Therearesiteswithminimalrecombinationinthegenome!</p><p>Positionalcloning</p></li><li><p>Mutagenesis+Wholegenomeseq</p><p>Zebrafish MutationProject(ZMP)</p></li><li><p>ProblemswithZMP</p><p>1. Lowdepthofsequence Sequencinggametesislesssensitive</p><p>2. Difficultyinrecoveringfoundmutations,husbandry</p><p>3. Spaceconstraints</p></li><li><p>Positionalcloning+Seq:Abetterapproach?</p><p>Obholzer etal.,2012</p></li><li><p>4.DegreeofSaturationA.Howefficientisthemutagenesis?</p><p>Mullinsetal.,1994B.Howmanyhitsonthesamegene?</p><p>Alsodependsonthegenesize</p></li><li><p>5.ProofofCandidateGenes</p><p>1. Incaseofmultiplealleles:complementation2. Rescueassays3. Morpholino4. Reversegenetics</p></li></ul>