X-chromosome inactivation in differentiating mouse embryonic stem cells carrying X-linked GFP and lacZ transgenes
Original Article | Published: 1 September 2004
Shirin Farivar1, Shinpei Yamaguchi1, Michihiko Sugimoto1,3 and Nobuo Takagi*,1,2
1Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan,
2Hokusei Gakuen University, Sapporo, Japan
and 3BioResource Center, Riken Tsukuba Institute, Ibaraki, Japan
Three new female ES cell lines (GLM1, GLP1 and GLP2) were established from mouse embryos carrying GFP (green fluorescent protein) and HMG-lacZ transgenes on one of two X chromosomes
in cis. Using these cell lines, we studied the temporal relationships among three events relevant to X-chromosome inactivation: replication asynchrony of the X chromosome, and quenching of GFP fluorescence and β-galactosidase (β-gal) activity, during cell differentiation induced by embryoid body (EB) formation and retinoic acid (RA) treatment. In embryoid bodies adhering to the bottom of culture dishes, GFP-negative cells appeared first in the peripheral outgrowths 4 days after the initiation of EB formation, followed about 24 hours later by the appearance of cells negative for β-gal and those having a single allocyclic X chromosome.
Although the frequency of cells with an allocyclic X chromosome could reach 80% in adherent
embryoid bodies, it tended to remain low and variable in embryoid bodies maintained in suspension. In spite of apparently parallel extinction of GFP and lacZ in embryoid bodies, their concurrent occurrence did not always characterize RA-induced differentiation. Moreover, an allocyclic X chromosome was identified in not more than 20 percent of informative metaphase cells up to 10 days after initiation of RA treatment. These findings suggest that RA-induced differentiation of female ES cells does not always accompany X-inactivation.