Generation and Characterization of a Novel Mouse Embryonic Stem Cell Line with a Dynamic Reporter of Nanog Expression
Figure 5
Nanog expression in FACS-sorted Nd ES cells.
(A) Representative histogram of FACS-sorted Nd sub-populations, grown in serum/LIF. VNP-low (VNPL) and VNP-high (VNPH) populations were collected for posterior analysis. (B) Nanog:VNP expression after re-plating sorted populations of Nd ES cells in serum/LIF. After 2–4 days, normal levels of heterogeneity are re-established, and expression of Nanog:VNP is similar between the three populations, either derived from the sorted VNPL and VNPH subsets, or from the whole population (“All”). Representative dot blots for FC analysis of VNP in non-fixed cells are presented in Figure S4. Statistically significant differences (p-value <0.05) observed between “All” and VNP sub-populations are denoted with (*), while statistically significant differences between VNPL and VNPH are denoted with (**). (C) mRNA expression analysis by RT-PCR of cells collected immediately after sorting (day 0) and four days after re-plating (day 4). The purified VNPL subpopulation (immediately after sorting) shows lower Nanog mRNA levels and higher expression of lineage-affiliated genes (Fgf5, Gata6 and T-brachyury). After culture for 4 days, both VNPL and VNPH subsets show similar Nanog mRNA expression. Expression of lineage markers in the VNPL subpopulation is still more elevated after 4 days of culture, most likely reflecting the slower reversion to heterogeneity (Figure S4). Similar levels of Oct4 and Sox2 expression are observed for all analysed samples.