les8 - Previous lesson 1) From ES cell to mouse 2)...

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Unformatted text preview: Previous lesson 1) From ES cell to mouse 2) Targeting vectors for HR PSM NSM Left Homology Right Homology Examples Ex 1 mouse M33 gene : Nature 1998 exon 5 : homol. seq. with Polycomb ( D. melanogaster ), Xenopus seq. → strongly conserved → KO of exon 5 generated see fig. : T vector without NSM but via poly (A) capture system selection via Eco RI southern:- by EcoRI site in neo R shorter than wt- by neo R incorp in mRNA v 2-5 kb longer- Western: no protein- 197 neo R clones 17H recombinants ( ⇒ 10% is a lot)- 7/17 clones selected → chimera’s- 4/7 germline transmission- cross -/+ x -/+ →-/- +/- +/+ in 1/2/1 ratio = OK- age ∆ 4w : die with colon /teeth problems- largest part XY -/- males had female characteristics (clitoris, vagina, hermaphrodites,...) M33-/- leads to “sex reversal” Ex 2- PNAS 1990 : c-fyn oncogen- LH 5.5 kb- SH 0.8 kb- promotor-trap neo R c-fyn expr. in ES cells - poly-A-trap NSM DT-A- fig- also ATG mutated → only expr. from neo R ATG and BamH1 new- only after HR : neo R + DT-A R → OK- bij integr. in trancr. active site : neo R + DTA S → +- bij integr. in tr. silent site : neo S → +- [ DT-A blocks ADP-ribosylatie at EF-2 ] ex 3 : mutants generated to mimic human pathology 2. CF pathology obstructed airways obstructed gastro-intestinal channel obstructed genital ducts CFTR gene found in 1990 : ‘CF transmembrane conductance regulator’ → defect in cAMP mediated Cl- ion-transport CFTR-/- mouse very hard → finally by exon 10 KO → lethal ± 4w- intestinal obstructions- perforations + lethal infections = typically CF- but NOT problems reprod. channels, pancreas, hepatocell., respir. problems) mouse model partially valid ex 4 : mutants generated to mimic human pathology hprt ko Lesch-Nyhan syndrom: - but hprt ko no pathology /phenotype- ‘fundam ≠ mouse-man for function this gene’?- Realize: mice not just = little men ex 5 GT by HR → KO : can be lethal/ lead to sterility many examples 1) TF = tissue factor- TF-/- lethal by angiogenesis problems- → TF role in angiogenesis - also TF -/+ lethal ! 2) CREM (cAMP responsive element modulator)-/- alive, but sterile →-/+ x -/+ breeding Need for methods to introduce: subtle mutations tissue specific mutations inducible mutations 6.6. “Classical” way to introduce subtle mutations in mice via HR 6....
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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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les8 - Previous lesson 1) From ES cell to mouse 2)...

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