3BA8EB8Cd01 - Chapter 8: Random-mutagenesis 8.1....

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Unformatted text preview: 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 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 ... 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 2) example 2: Ods- Ty minigene in FVB/N mice - a certain Tg line:- degeneration eyes- 0/1000 offspring D 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 ) - 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 3) example 3: ducky mutation- insertion mutagenesis also spontanously, 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 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 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 1. Both via plasmids tranfected in ES cells > transposition frequency low 2. Inject in zygote2....
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This note was uploaded on 05/30/2010 for the course WE CHBIBI0000 taught by Professor Krisvleminckx during the Spring '10 term at Ghent University.

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3BA8EB8Cd01 - Chapter 8: Random-mutagenesis 8.1....

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