Expression of GFPcre in fetal liver cells of ErGFPcre mice. (A) Immunoblot analysis of FLCs. Littermates hemizygous GFPcre positive (–/+; ki) or GFPcre negative (–/–) and wild type at the EpoR (WT) were isolated at 13.5dpc and genotyping was performed by multiplex PCR on yolk sac DNA (top). Cell lysates of FLCs were immunoprecipitated with an anti-GFP antibody and analyzed by immunoblotting with an anti-Cre or anti-GFP antibody (bottom). (B) Analysis of FLCs by flow cytometry. FLCs derived from wild-type (i), hemizygous (ii), or homozygous (iii) ErGFPcre embryos at E12.5 were analyzed for GFP fluorescence. Data were plotted as relative fluorescence intensity versus the cell number. The genotype of the embryos used for FLC preparation was determined by multiplex PCR analysis on yolk sac DNA (inset). (C) Analysis of FLCs by fluorescence microscopy. FLCs derived from wild-type (Balb/c) or hemizygous ErGFPcre (ErGFPcre) littermates at embryonic stage E13.5 were cultivated in 0.8% methylcellulose supplemented with 0.5 U/mL Epo and 5 mM Hoechst 33258. After 24 hours, cells were analyzed for Hoechst and GFP fluorescence using fluorescent microscopy. The merged pictures were obtained by overlaying the corresponding phase contrast, Hoechst, and GFP images (scale bar, 10 μm).