les5 - 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 introduce more genes at once (e.g. cluster) introduction large genes Most genes: 20-200kb still limited eg DMD gene, E-Cadh 2000 kb/giant 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 : expression and phenotype + study regulatory sequences by generating TG mice with truncated P1 constr Expression? Regul. seq. from 15kb 5’ to 5kb 3’
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disadvantages - single copy plasmids big cultures of E. Coli needed (500ml) 100μg plasmide DNA - big fragment separate via PFGE (FIGE) dialyse versus TE/spermine/spermidine/NaCl (to reduce sheering forces)
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BAC - 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 see figure - with ORI, rep,. .., CM R of F plasmid - with NotI, T7, SP6, LoxP - NotI site good to determine size of insert - 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 mutation over-expression gene cluster study regulatory sequences
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1) Zipro1 gene = encodes zinc-finger transcription factor - Zipro1 % mice : no phenotype (by complem. by another transcription factor?) - Zipro 1 tg BAC mice : have a phenotype zipro 1 essential for development embryo - 4 founders : - 1-14 copies zipro - The more zipro -genes the more phenotype Proliferation of skin and cerebellum (+ bold phenotype) - Zipro 1 tg BAC with mutant Zipro : no phenotype (additional advantage BAC: to 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 ritm unknown regulation in mammals/ in hypothalamus In lower organisms number of mutants period ”, timeless ”, “ frequency = “ clock ” mutants with cycle of 27 hours iso 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
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This note was uploaded on 05/28/2010 for the course WE BIBI000000 taught by Professor Johangrooten during the Spring '10 term at Ghent University.

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les5 - Today: P1, BAC, YAC transgenesis Inducible...

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