88A29C26d01 - Previous lesson 1) From ES cell to mouse 2)...

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Previous lesson 1) From ES cell to mouse 2) Targeting vectors for HR PSM NSM Left Homology Right Homology
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Early years Mix of types of targeting vectors - No NSM - No NSM but PSM with promotor trap or polyA trap - Combinations of promotor trap PSM and polyA trap NSM - PSM/NSM Later - Almost exclusively PSM/NSM
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Examples Example 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
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- 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”
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Example 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
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- 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 ]
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Usually PSM/NSM Some examples
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Usually PSM/NSM Some examples
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Usually PSM/NSM Some examples
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Usually PSM/NSM Some examples of lack of NSM
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Usually PSM/NSM Some examples of lack of NSM
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Further examples: mutants sometimes generated to mimic human pathology Usually successful Sometimes problematic 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
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CFTR-/- mouse very hard to obtain finally succeeded 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 (hypomorphic allele or fundamental difference mouse-human gene function or pathology)
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Other example: mutants generated to mimic human pathology hprt ko Lesch-Nyhan syndrom: (X-liked disease/mental retardation/automutilation/aggression. ..) - HPRT deficieny and strong genetic link in humans - but hprt ko mouse no pathology /phenotype - ‘fundam mouse-man for function this gene’?
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88A29C26d01 - Previous lesson 1) From ES cell to mouse 2)...

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