Figure 3.

Experimental approaches for the detection and manipulation of the core TSC maintenance factors Eomes and Cdx2, followed by gene expression profiling. Three complementary experimental approaches were chosen for expression profiling of (a, b) TSCs and (c) mES cells with inducible expression of Eomes or Cdx2. (a) For the expression profiling of bona fide TSCs and early stages of differentiation, Eomes ^GFP TSCs were FACS-enriched for GFP^high cells and forced towards differentiation by withdrawal of stemness maintaining conditions for 3d. (b) For the identification of Eomes-regulated target genes, TSCs harbouring Eomes conditional alleles (Eomes ^CA) in combination with a tamoxifen-inducible Cre-estrogen receptor allele (R26Cre^ER) were used for conditional inactivation in vitro. Cells were tamoxifen-treated for 24 h and (b′) Cre-mediated excision was monitored by genotyping PCR, and (b′′) presence of EOMES protein was analysed by Western blot. (c) To transcriptionally profile the initiation of Eomes- and Cdx2-induced target genes during ES cell to TSC conversion, mES cells with doxycycline-regulated expression were induced for 48 h. (c′) The expression of V5-tagged EOMES or CDX2 protein was monitored by Western blot. MEF, mouse embryonic fibroblast; TSC, trophoblast stem cell; asterisks (∗) indicate the different time points of gene expression profiling by gene arrays.