Selective ablation of differentiated cells permits isolation of embryonic stem cell lines from murine embryos with a non-permissive genetic background.
Embryonic stem (ES) cells enable the engineering of precise modifications to the mouse genome by gene targeting. Although there are reports of cultured cell contributions to chimaeras in golden hamster, rat and pig, definitive ES cell lines which contribute to the germline have not been demonstrated in any species but mouse. Among mouse strains, genetic background strongly affects the efficiency of ES isolation, and almost all ES lines in use are derived from strain 129 (refs 1,4,5) or, less commonly, C57BL/6 (refs 6-8). The CBA strain is refractory to ES isolation and there are no published reports of CBA-derived ES lines. Hence, CBA mice may provide a convenient model of ES isolation in other species. In ES derivation it is critical that the primary explant be cultured for a sufficient time to allow multiplication of ES cell progenitors, yet without allowing extensive differentiation. Thus, differences in ES derivation between mouse strains may reflect differences in the control of ES progenitor cells by other lineages within the embryo. Here we describe a strategy to continuously remove differentiated cells by drug selection, which generates germline competent ES lines from genotypes that are non-permissive in the absence of selection.
McWhir
J
J
Roslin Institute, Department of Molecular Biology, Midlothian, UK.
Schnieke
A E
AE
Ansell
R
R
Wallace
H
H
Colman
A
A
Scott
A R
AR
Kind
A J
AJ
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D002678:Chimera / Q000235:genetics*; D003433:Crosses, Genetic; D004351:Drug Resistance; D004622:Embryo, Mammalian / Q000166:cytology; D005260:Female; D005839:Gentamicins / Q000494:pharmacology; D005854:Germ Cells; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA / Q000196:embryology*; D008822:Mice, Transgenic; D013234:Stem Cells / Q000166:cytology*
D000900:Anti-Bacterial Agents; D005839:Gentamicins; C010680:antibiotic G 418