1C4F1F99d01 - Chapter 8 Random-mutagenesis 8.1 Introduction...

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Chapter 8: Random-mutagenesis 8.1. Introduction EST gDNA KO mutant cDNA TG overexpression = gen(otyp)e to phenotype = reverse genetics - up until recently only few sequences available - mutagenesis of all genes by TG-KO: impossible (however…) - but many mutants needed (even several/gene) saturation of genome by random mutagenesis phenotype if interesting identify gene = phenotype genotype = forward genetics
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8.2. Insertion-mutagenesis - ± 5% of transgenic DNA insertion mutation - mutations insertions but also deletions - not understood - difficult to identificy mutated (deleted) gene - usually recessive - several examples in literature (lethality, extremities, stature, fertility...) - examples: steel, pygmy, hotfoot, extratoes, downless , limb deformation ...
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1) example 1: pygmy (Nature 1995) - Ty transgene in FVB/N (Ty gives grey colour to albino mice, acts as a mutagen and a “sequence tag”) pygmy phenotype = dwarphism - detailed study of mouse mutant > gDNA > BAC/P1 library > hybr. with TgcDNA > BAC clone > compare with WT deletion 56kb near Hmgi-c gene - pygmy mutant, no hmgi-c mRNA - hgmi-c-KO mouse also pygmy phenotype = confirmation
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2) example 2: Ods (“Odd sex”) - Ty minigene in FVB/N mice - a certain Tg line: - degeneration eyes - 0/1000 offspring though 50% XX 50% XY - testis TG XY normal TG XX small, normal Sertoli/ Leydig structures - FISH analysis of mutant Ty gene on distal chr 11 (nearby sox9 )
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- TG mouse gDNA BAC library : screen with Tyr ase gene BAC clone 200kb - contains Ty gene + sox9 gene - Tyrosinase insertion in regulatory gene that determines expr. of sox9 normal XX : regulator represses sox9 expr. normal XY : regulator inactive due to repressor no repression sox9 mutant XX : regulator inactivated by insertion no repression of Sox9 expression Sox9 male
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3) example 3: ducky mutation - insertion mutagenesis also spontaneously, in lab by retrovirusses / transposons - mouse colony spontaneous mutant ducky - = mouse epilepsy (EEC, ataxia, seizures, brain pathology) - sterile + lethal at 5w age - mutation by retrov. integr. in intron of du gene fusion transcript du -retrovirus - also some normal transcripts by partially neglecting of viral SA signal
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4) example 4: MRL lpr/lpr mouse - AIC after immune response by Fas-FasL system - mutant: only 5% of normal expression of Fas by integration transposon in Fas gene lymphoproliferation (lpr) - transposon contains poly(A) signal truncated Fas mRNA in 5% of cases transposon poly(A) neglected > 5% full length Fas transcripts
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Transposons - Now used systematically for insertion mutagenesis - Many examples: Minos Tol2 PiggyBac Sleeping Beauty - Sleeping Beauty : from Tc1/mariner group: Tp of fish, inactive in mouse - Awaken as “sleeping beauty”: Tp mutant that is active in mouse - Always 2 components system: Transposon itself (IR----cargo----IR) Transposase
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1. Both via plasmids tranfected in ES cells > transposition frequency low 2. Inject in zygote of pCAGG-GFP Tp - Several animals - Cross with transposase -TG - In F1: Transposon and Transposase meet each other after fertilization:
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