les7 - Last time: ES cells- derived from ICM of...

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Unformatted text preview: Last time: ES cells- derived from ICM of pre-implantation blastocysts- real stem cell: can differentiate in all types of cells, also germ cells- can be kept in culture (in undifferentiated status)- can be manipulated (transfected, antibiotics selection,…)- from many species, but only in mice…- after that injectable in host blastocyst > chimeric embryo’s and mice This lesson: 1. ES > mice 2. Genetic manipulation of ES cells via HR 6.2. cells → mice- genetic manip. ES cells : usually aim is generating mutante mouse- via introduction ES in existing embryo (“ES on its own can not develop to new mouse”, host embryo needed )- ES cells can take part in development- hope: ES → germ cells in chimeric mouse +/+ +/- BALB/c (e.g.) 129- 1984 : ES → chimeric mouse- XY ES cells introd. in XX embryo : advantages : 1. XY cells form germ cells with high % (repression of XX germ cell formation) 2. XY chimera → fast expansion colony- source of ES cells (strain) important for degree of chimerism, germline transmission and for detection chimera’s- e.g. ES of 129/J mice introd. in blastoc. of C57BL/6 → ES cells dominate development (+ advantage simple selection chimera’s : agouti + black)- also 129/J ES + BALB/c blastocyst (agouti on white) or 129/J ES + SWISS blastocyst (agouti on white) introduction of ES in host embryo 1) injection in blastocoel- not simple- expensive microinjection device needed- damages blastocyst- reliable 2) aggregation : ES + 8 cell morulae ( ← ZP removed) overnight then transfer in uterus of pseudo-pregnant female birth chimera’s- 3w after transfer- identify coat color : very simple (e.g. agouti on albino or agouti on black)- % chimerism varies strong (5% - 95% - 100%)- starting from XY ES cells → many “ ”chimera’s often also hermaphrodites (small testis, ovotestis, urogen. abnormalities) after identif. of chimera’s : test if mutation in germline 1. chimera x wt (albino) mouse WT albino heteroz. agouti WT agouti 2. heteroz x heteroz 25% homozygotes attention : chimeric : half of sperm cells mutant (cf mut. ususally heteroz. in ES cell) also chimerism within genitals Realize: mixed genetic background by intercross -/+ advantage: coat colour selection ES individuals disadvantage: mixed background (129 x BALB/c) alternative?...
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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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les7 - Last time: ES cells- derived from ICM of...

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