1OverviewModels in laboratory researchThe unique position of the mouse as a modelMouse genetics 101Genetic engineering for hypothesis testingGenetic variationWhy Use Models?Allows for controlled experimentsEnvironmental variables can be controlledDosage or exposures can be controlledExperiments can be replicated2GenesPhenotypesGenesEnvironmentEnvironmentStochasticProcessesChapel HillGenesEffects of Genes Can Be ComplexGenePhenotypePhenotypesGenesPleiotropyHeterogeneitygataccagattagtagttagagttgagagtccgctagatcgcGene ContentDETECTIONGeneticToolsMutagenesisGeneticsUnique Position of the MouseBiologicToolsHistologyMolecularProfileProteomeContentStrain: A/JID: XXXXTissue: colCHARACTERIZATIONPhysiologyEngineeringToolsVALIDATIONTransgenicsKnockoutsKnockins3Why Mice As an Experimental Organism?Short life cycleEasily bredHigh fecundityHardyRequires little spaceLarge amount of phenotypic variationEasy to genetically engineerMammalian speciesEvolutionary Relationships0 myr bp1002003004005006007008009001000C. elegansD. melanogasterXenopusMiceHumansThe MusSpecies Groupdomesticusmusculuscastaneusbactrianusmacedonicus(Greece>Iran>Israel)spicilegus(Austria.Ukraine>Bulgaria)spretus(Spain/N. Africa)caroli(Indonesia)cookii(India/S.E. Asia)cervicolor(Nepal/S.E. Asia)booduga(India/Burma/Pakistan)dunni(India/Sumatra)TheM. musculusgroup6.0 3.05.0 4.0 2.0 1.0 0 myr bp4Genetic StocksOutbred• segregating many alleles• ex, Swiss mice, Wistar ratsHybrid• isogenic until bred• ex, B6D2, B6SJLInbred• genetically identical (isogenic)• ex, C27BL/6JMutant/Engineered• specific defects• may or may not be ‘genetically clean’Exercise AYou have developed a compound that you think will help to prevent rejection of transplanted hearts, and you want to test this experimentally.The experiment will involve heart grafts between a donor and recipient mouse (whose own heart is not removed) with a control group and one treated with the compound.The following mouse strains are available: outbred ICR and CD1 and inbred C57BL/6J, A/J, and FVB/NJ.Which strains will you use as donor and recipient? Why?Exercise BYou also need to test the potential toxicity of the compound, and will want to do a long-term study with control and treated mice. You know it is not acutely toxic.Being a toxicologist, you reason that in this case since you wish to model humans who are genetically heterogeneous, you decide to use outbred genetically heterogeneous ICR mice, the strategy used by virtually all toxicologists.Do you decide to go with your initial intuition? Why?5Identify the ‘Experimental Unit’The unit of randomizationTwo experimental units must be capable of being assigned to different treatmentsCould be:a cage of animalsa single animalan animal for a period of timea well in a tissue culture dishThe Problem With Genetic HeterogeneityTreated Controlbeagle chickenmouse crowhorse froggerbil hamsterlion beavercat dograbbit toadA Better DesignTreated Controlbeagle beaglemouse mousehorse horsegerbil gerbillion lioncat catrabbit rabbit6A Better DesignTreated ControlC57BL/6J C57BL/6JA/J A/JFVB/NJ FVB/NJDBA/2J DBA/2JSWR/J SWR/JSJL/J SJL/JBALB/cJ BALB/cJVariable Results With Heart Transplants‘We transplanted hearts of young … ICR into … recipient CD1. An outbred strain was selected since such animals are usually heartier and easier to handle …We are puzzled by our results … palpable heart beats were evident in the saline group long after acute rejections … were expected … Results in the experimental groups varied considerably …’Exercise C: Power Calculations for Sample SizeBarbiturate sleeping timeStrain Mean Std. Dev.BALB/c (inbred) 40 4ICR (outbred) 40 15What sample size would be needed to detect a 10% change in mean, with a 90% power and 5% significance level using a 2-sample t-test?Data from Jay (1955) Proc Soc exp Biol Med 90:3787Power Calculations for Sample SizeBALB/c (inbred) 23ICR (outbred) 2978Types of Genetic CrossesCross Matings UsesBackcrossA/a X A/AA/a X a/alinkage analysis; production of congenicstrainsIncrossA/A X A/Aa/a X a/aMaintenance of aninbred strainIntercrossA/a X A/aLinkage analysisOutcrossA/A X a/aa1/a2X a3/a4Initial step in strain production and linkage analysis; production of F1hybridsMaking an Inbred Strain% Homozygosity0 50 10098.7%F20InbredF1PF2Independent AssortmentInbred Strain20 or more generation of brother x sister matingIsogenicHomozyogusPhenotypically uniformLong-term stabilityUnique strain characteristicsInternational distributionEasily identifiable‘immortal clone of genetically identical individuals’9‘The introduction of inbred strains into biology is probably comparable in importance with that of the analytical balance into chemistry’Gruneberg (1952)Outbred StocksWidely usedCharacteristics not widely understoodAdvatages• cheap• easily available (no alternative for some species)• breed well• outbred like humans (?????)Disadvantages• unknown genetics (heterozygosity)• subject to genetic change (inbreeding, drift, selection)• lack of reliable background information• genotype not internationally distributed• not histocompatible• not easily identifiableCF1ICRCD1MF1Swiss-WebsterOutbred MiceB6D210Engineered ModelsAllows controlled experimental testing of• specific genes• specific environmental conditions or exposuresIdeally suited to test specific hypothesis generated from human population studies or other laboratory findingsEngineered ModelsTransgenics• usually used to over-express genes• can be global or tissue-specific• can be temporally regulated Knockouts/knockins• usually used in inactivate genes• can be global or tissue-specific• can be temporally regulated• can introduce genes into a foreign locus• can make amino acid modifications TerminologyTransgenic(carries foreign DNA;may or may not be mosaic;two parents)Mosaic(may or may not carry foreign DNA;two parents)Chimeric(may or may notcarry foreign DNA;more than two parents)11Mosaic vs Chimeric ProgenyMosaic ChimericPre-implantation Mouse DevelopmentFertilizationActivation of embryonic genomeCompactionBlastocoelic fluid accumulationTE and endoderm differentiationE0.5E2.5E3.5E1.5Transgenic ProductionFlush fertilizedoviduct E0.5pro-nuclear stageDNArecoverpro-nuclearfusionimplant intopseudopregnant femalestest for germlinetransmissionfoundertest for expressionand phenotype12Gene Targeting in ES CellsAnalyze offspringfor phenotypeelectroporateselectionanalyzecoloniestest