Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Adding a gene: Producing Transgenic MiceProduction of Transgenic MiceSlide 9Slide 10Slide 11Technique for Gene TargetingNow you have heterozygous ES cells--how do you make a homozygous mutant mouse?Now you have a chimeric mouse…Sometimes the effects are dramatic!Morphological Analysis of Bmp7 Knockout MiceSlide 17Slide 18Slide 19The Homeotic genes in DrosophilaSlide 21Slide 22Slide 23Slide 24Partial transformation of the first lumbar vertebra into a thoracic vertebra by knockout of the Hoxc8 geneSlide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Mutations in Pax3 lead to Waardenburg Syndrome I.Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Mutations in MITF lead to Waardenburg Syndrome II.Model organisms: mice• vertebrates!• mice are ~ 3 inches long, can keep many mice in a room.• generation time is ~ 3 months, so genetics can be done• history - scientists have worked with mice for 100 years• genetic tools - can introduce extra genes or remove a specific gene, then study the effect on development• Disadvantages: development inside the mother, hard to see. Expensive!Large Genome = 3 GbThe mouse provides a superb model for human development and disease becausewe share virtually ALL of our genesand use them in similar waysFigure 1.22Kit geneGenetic analysis: creating transgenic miceProblem: Find a cell line that can grow in tissue culture but also retains the potential to become part of a real embryo. Solution: Embryonic stem cellsEmbryonic stem cells:blastocyst-stage cells(from inner cell mass)that have been coaxedinto growing in cultureblastocystinner cell massBlastocyst stage cells can be easily incorporated into a different blastocyst stage embryo, allowing production of chimeric miceFig. 8.26mouse with 4 parents!!mom and dad have white furmom and dad have black furA mouse with3 of its parents(6 total!)Fig. 8.26Adding a gene: Producing Transgenic MiceProduction of Transgenic MiceEmbryonic stem cells (ES cells) are then incorporated into blastocysts, with the hope that they “go germline”. If so, a line is createdProduction of Transgenic MiceProduction of Transgenic MiceRNAGene XRNAGene XA normal cell has two copies of a gene (ie. BMP7) No RNAGene XRNAGene XNeo resistance gene1. Insert gene for resistance to the drug neomycin into the middle of gene X, destroying its function. (Gene X is contained in a DNA plasmid.)2. Introduce gene X KO plasmid into ES cells and use homologous recombination to replace one of the wildtype copies of gene X with mutant gene. Recipe to "knockout" a gene: Mario CappechiOliver SmithiesTechnique for Gene Targeting#1#2#3Now you have heterozygous ES cells--how do you make a homozygous mutant mouse?#4#5Now you have a chimeric mouse…#6#7Sometimes the effects are dramatic!BMP7 knockoutWild-typeMorphological Analysis of Bmp7 Knockout MiceSometimes the effects are not dramatic--no phenotype!Mouse models of human diseaseallow us to design and test new treatmentsOliver SmithiesCFTR and cystic fibrosisUltrabithorax mutantWildtyperemember me?The Homeotic genes in DrosophilaFig 6.35ANT-C BX-CEd Lewis had predicted that the homeotic genes would shape the body plans of all animalsIn vertebratesthe Hox genes have been duplicated,creating four clustersFigure 8.30Different Hox genes are expressed at different places along the anterior-posterior body axisKnocking outHoxc8Figure 8.30Partial transformation of the first lumbar vertebra into a thoracic vertebra by knockout of the Hoxc8 geneGenetic analysis of Hox genes is more complicated in miceparalog groupKnocking outHoxa10, Hoxc10 &Hoxd10Figure 8.30The duplication of the Hox clusters means that in the mouse, Hox genes work togetherto give each body region its own identityLumbar vertebrae transformed to thoracic vertebraeFigure 8.32wildtypeHoxa10 Hoxc10 Hoxd10triple mutantRemember the segment-polarity genes wingless and engrailed?WgEnRetroviruses can also cause cancer by inserting next to and thusactivating the expression of proto-oncogeneswnt-1 geneexonsTranscribe to mRNA5 kilobasesretroviral insertionsites in different tumorsExpresses Wnt-1Expresses En-1Wildtype brainWnt-1, The mouse homolog of wingless,is normally expressed at the midbrain-hindbrain junctionExpresses Wnt-1Expresses En-1Structures lost in Wnt-1 mutantWildtype brainBrain of Wnt-1 mutantRules of EvidenceWhat type of experiment is this?Pax6Normal eyeAniridia eyesmall eye mutant mousePax6 regulates eye development in flies, squid, mice, and usirisno irisWhen eyeless (Pax6 homolog) is expressed at the ends of fly legs, extra eyes form there!When squid Pax6 homolog is expressed at the ends of fly legs, also see extra eyes!ectopic eye (squid Pax6)ectopic eye (fly Pax6)Wild-type Splotch mutantThe Pax-3 gene is altered in a classic mouse mutationMutations in Pax3 lead to Waardenburg Syndrome I.• dominant mutation• eyes can be different colors• white patch of hair (forelock)• deafnessWhy Models MatterThe Example of Mutation of the Kit gene in humans and mice“Piebaldism” • Affected individuals are anemic, sterile, deaf, and lack pigment in certain skin cells• Kit encodes a receptor tyrosine kinase and is required for cell proliferation in neural crest, blood, and germ cellsFigure 1.22White spotting and Steel:Connecting classic mouse mutations to stem cells and cancerAn example of stem cells: the blood cell lineageCells lacking signal behave differently than cells lacking receptor +++mutant+++++++mutantmutant++++++++++++++ If mutant cells lack signal, they can be rescued by wildtype neighbors which make signal.If mutant cells lack receptor, they cannot be rescued by wildtype neighbors which make signal.+++mutant+++++++mutantmutant++++++++++++++Thanks, I needed that!What? I can't hear you!Mosaics can help us understandgene and thus protein functionWhite-spotting and Steel: Which is signal and which is receptor??The mutant blood cells migrated to the bone marrow.Experiment #1Put blood cells from Steel homozygous mutant embryos into a wild-type host.Experiment #2Put blood cells from White-spotting homozygous mutant embryos into a wild-type host.These mutant blood cells did not migrate to the bone marrow.Steel is the Signal- Mutant cells can still receive informationWhite-spotting is the receptor- Mutant cells cannot receive informationSteel encodes a diffusible ligand and White-spotting(Kit) encodes its
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