Fig. 2. Generation of GFP-Bry ES cells. (A) Schematic structure of the mouse brachyury gene locus and the targeting vector. LoxP sites are indicated by black triangles on either side of the Neo gene. The hatched box indicates an exon derived from plasmid pBK-CMV, which contains the SV40 polyadenylation signal. TK: Herpes simplex thymidine kinase gene. H: HincII. (B) Blast-CFC potential: EBs from both ES cell lines were harvested on the indicated days of differentiation and cells assayed for BL-CFC. Colonies were scored after 4 days of culture and were referred to as 2nd EB (secondary embryoid bodies) and Blast (blast colonies). Data are presented as the mean number of colonies from three dishes. Bars, where visible, represent s.e. of the mean. (C) Fluorescence microscopy of Bry-GFP EBs at day 3 of differentiation. Wild-type EBs did not show detectable levels of fluorescence (not shown). Photos were taken with a Hamasura camera at 100x magnification. (D) EBs from the GFP-Bry ES cell line were harvested daily between day 1 and 6 of differentiation. Cells were used for expression analysis of brachyury by RT-PCR using gene specific primers and (E) flow cytometric analysis for detection of the GFP protein.

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