Isolation of murine embryonic stem and embryonic germ cells by selective ablation.
The isolation of murine embryonic stem (ES) cells has been almost exclusively from the 129 mouse strain. Other mouse strains, such as CBA, have proven refractory to ES cell isolation by conventional means. We previously reported the isolation of 87.5% CBA ES lines by selective ablation of differentiating cells (McWhir et al., 1996). Here, we report the isolation of ES and EG cells from 94% CBA embryos hemizygous and homozygous for a neomycin-resistance transgene under the transcriptional control of the Oct3/4 promoter (Oct/neo). Since expression of the Oct/neo transgene only confers drug resistance to undifferentiated cells of the inner cell mass, selection results in the ablation of differentiating cells from the culture. The efficiency of ES isolation by selective ablation in homozygotes is twice that in heterozygotes. ES isolation frequency in permissive strain 129 embryos is enhanced by treatment with an inhibitor of the extracellular-signal-regulated kinase (ERK) pathway but this effect is not sufficient to permit ES isolation from the CBA strain.
Gallagher
Ed J
EJ
Roslin Institute, Roslin, Midlothian ETH 9PS, UK. ed.gallagher@bbsrc.ac.uk
Lodge
Peter
P
Ansell
Ray
R
McWhir
Jim
J
D000818:Animals; D015153:Blotting, Western; D019070:Cell Lineage; D015536:Down-Regulation; D004622:Embryo, Mammalian / Q000166:cytology*; D005854:Germ Cells / Q000166:cytology*; D048369:MAP Kinase Kinase 1; D051379:Mice; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology*
D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases