24DFA19Ad01 - Forgotten last time Diploid mutations in ES...

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Forgotten last time: Diploid mutations in ES cells - Via intra-chromosomal 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-/- mice 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 mutagenesis 7.1. The Cre-LoxP system - based on Cre recombinase and loxP sites of E. Coli phage P1 Allows the following: - non-selectable mutations/removal selection genes/ translocations/big deletions... - cell-type-specific and inducible mutagenesis (solution for problems with conventional full KO,...)= conditional mutagenesis - site-specific mutagenesis
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Cre-recombinase - 38kDa - C re gene (cyclization recombination) - Member λ integrase superfamily - Essential for phage P1 : make P1 genome cyclic - no co-factors needed, no specific DNA structures needed for recombination 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 - usually depicted as an arrow/triangle - depending on direction loxP : + possible results after cre inversion excission / integration translocation
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enzymatic reactions equilibria! E.g. after excission also integration possible! (can be applied) molecular event of recombination: - recombination by ssDNA break in core regio of loxP - interaction ssDNA with P-Tyr 324 of Cre - 4 Cre molecules needed for a recombination event at 2 lox P’s
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pointmutations in core lead to variant loxPs - still homotypic R possible - no longer heterotypic R possible - interessting perspectives (later) also mutant loxP that allow only integration, no excission
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for efficient recombination between 2 loxP’s: - minimum 82bp distance - maximum : no limit e.g. in literature: several Mb - 34bp = a lot probably no cryptic loxP-like sites in genome (?!) - based on mouse genome sequence: indeed no such sequences - however: see further protamine-cre
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Cre/loxP origin - Prokaryotic: cre has easy access to loxP’s (no nuclear envelop) - Eukaryotes: - Cre through nuclear pores - no problem - yet most cre molecules used have extra NLS
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- loxP sequences usually cloned in introns at least 150bp from SD/SA - loxP best not in controle regio’s : enh/ prom/ splice signals - 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 of ES cells by classical targeting vector (with homology/ 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 cross with Cre tg mouse recombination in vivo or 3) Or combination of both.
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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) Major application: conditional mutagenesis
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Classically Better: remove Neo R gene Even better
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2) “Non-selectable” mutations: e.g. pointmutation
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