les9 - Forgotten last time: Diploid mutations in ES cells -...

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Forgotten last time: Diploid mutations in ES cells - Via intrachromosomal HR - 1/100.000 - But much better in Bloom ES cells: 1/5.000 - Bloom ES: more mitotic recombination - Bloom = mutation in exon 3 Blm gene - Blm/Blm mice viable, fertile, increased cancer incidence: 5% of mice after 1y (compare p53% 100% after 7 months)
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System to control Bloom gene in WT ES cells: shut off WT Blm gene expression by DOX in ES cells:
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So: in ES cells:
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7. Conditional HR 7.1. The Cre-LoxP system - non-selectable mutations/removal selection genes/ translocations/big deletions. .. - cell-type-specific and inducible mutagenesis (solution for problems with conventional full KO,. ..) - based on Cre recombinase and loxP sites of E. Coli phage P1
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Cre-recombinase - 38kDa - C re gene (cyclization recombination) - Member of int family ( λ integrase superfam.) - Essential for P1 : make P1 genome cyclicP1 - no co-factors needed, no specific DNA structures needed for R at loxP sites works in mouse, yeast, bacteria, plants,. .. - temp opt = 37°C - target site is loxP (locus of x-ing over of P1)
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loxP - 34 bp long - 2 inverted repeats of 13 bp + core/spacer 8 bp - oriëntation spacer determines oriëntation lox P - fig - depd. direction loxP : + possible results after cre fig inversion excission / integration translocation
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enzymatic reactions equilibria! E.g. after excission also integration possible! (can be applied) molecular : - recomb. by ssDNA break in core regio - interaction ssDNA with P-Tyr 324 of Cre - 4 Cre molecules needed for a recomb. at 2 lox P’s
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pointmutations in core: - still homotypic R possible - no more heterotypic R possible - interessting perspectives (later) also mutant loxP that allow only integration, no excission
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for effic. recomb. between 2 loxP: - minimum 82bp - max : no limit eg. in literature: several Mb - 34bp = a lot probably no cryptic loxP in genome genome sequence: indeed no such sequences - however: see further protamine-cre
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Cre/loxP origin - Prokaryotic: easy access to loxP (no nuclear envelop) - Eukaryote: - Cre through nuclear pores - no problem - yet most cre molecules used have now NLS Realize: after R at loxP a loxP remains in genome
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So, for genetic manipulation : principle : - build in LoxP sequences in genome ES by TV with homologous seq / PSM / NSM - after selection correct ES cell: 1) Cre transfection in ES site-specific recombination in ES cell Mouse or 2) make mouse from ES cells, then x Cre tg recombination in vivo 3) Or combination of both.
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LoxP seq. usually intron at least 150bp from SD/SA loxP best not in controle regio’s : enh/ prom/ splice signals by cut with R enzyme + synth. LoxP cloning
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7.2. First applications : 1: Removing selection genes 2: Non-selectable mutations Later applications - deletions - gene replacement - translocations - integrations - promotor studies (via floxed STOP-reporter mouse) Conditional mutagenesis
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2) Non-selectable mutations: eg. pointmutation tk
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les9 - Forgotten last time: Diploid mutations in ES cells -...

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