A2BF377Ad01 - Today P1 BAC YAC transgenesis Inducible...

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Today: P1, BAC, YAC transgenesis Inducible transgenesis Some application areas ES cells (intro, differentiation)
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5.7. P1, BAC, YAC transgenesis need for over-expression of large, stable constructs more success for introduction of all regulatory sequences less dependent of chromosomal position effects (LCRs) introduce more genes at once (e.g. cluster) introduction large genes Most genes: 20-200kb but some… eg DMD gene, E-Cadh : 2000 kb/giantic introns
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P1 inserts: max 100kb E.g. - hApo-B gene = 45kb - hApo-B cDNA tg mouse : never successful to get expression - P1 -hApo-B tg mouse : full expression and nice phenotype + study regulatory sequences by generating TG mice with truncated P1 constructs Expression? > Regul. seq. from 15kb 5’ to 5kb 3’
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disadvantages PACs - single copy plasmids large cultures of E. Coli needed (500ml) 100μg plasmide DNA - big fragment - separate via PFGE (FIGE) - dialyse versus TE/spermine/spermidine/NaCl (to reduce charge of polyanionic DNA > reduce sheering forces)
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BAC inserts - since 1992 - inserts of ± 200-300kb - in E. Coli / stable : based on F plasmid - single copy - many libraries available (PCR screening) - eg pBELO BAC 10-11 - with ORI, rep,..., CM R of F plasmid - with NotI, T7, SP6, LoxP - NotI site good to determine size of insert (GCGGCCGC) - SP6, T7 good for end sequencing insert - Not I digest fragment via PFGE, dialysis, injection - Usualy 1-5 (seldom more) injected copies / zygote - Can also be used in ES cells
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Examples Creating a phenotype complementation of a mutant phenotype (pinpointing gene) over-expression gene cluster study regulatory sequences
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1) Zipro1 gene = encodes a zinc-finger transcription factor - Zipro1 % mice : no phenotype (by complem. by another transcription factor?) - Zipro 1 tg BAC mice : have a phenotype (proliferation of skin and cerebellum + hairless) zipro 1 essential for development embryo - 4 founders : - 1 to14 copies of Zipro - The more Zipro -genes the more phenotype - Zipro 1 tg BAC with mutant zinc finger: no phenotype (additional advantage BAC: can be modified in E. Coli)
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2) To revert a mutant phenotype by complementation identification of mutated gene - Via ENU mutagenesis (Cell, 1997): Mutant with changed day-night rhythm unknown regulation in mammals/ in hypothalamus In lower organisms number of mutants period ”, timeless ”, “ frequency = “ clock ” mutant with cycle of 27 hours instead of 24 h - Via backcross : clock gene on chr.5 between D5Mit 307 and D5Nwu2 (see figure )
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- BAC contig - Tg BAC mice BAC’s introduce in clock/clock background (by crossing!!) + study day-night rhythm (see figure)
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