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Fig. 5. GDF3 reduction of function through AD0857 genetrap in mouse embryonic stem cells. (A) Diagram of mouse Gdf3 genomic locus containing the AD0857 genetrap. The Gdf3 prepro domain (exon 1 and most of exon 2) is shown in red, GDF3 mature (exon 2) is shown in green, and the genetrap insertion is shown in blue. The RACE Tag for the genetrap contains most of exon 1 of GDF3. (B) Western blot of wildtype (WT) and AD0857 genetrap (GT) ES cells with p{alpha}GDF3 (top panel, prepro GDF3; middle panel, mature GDF3) and m{alpha}tubulin (bottom panel). (C) Morphology of WT and GT ES after 4 days of culture in the presence or absence of LIF (x10). (D) RT-PCR of WT and GT ES after 4 days of culture in the presence or absence of LIF. FGF5 is a marker of pluripotent epiblast. BU is a marker of mesoderm. SOX2 and OCT3/4 are markers of the undifferentiated state. ß-actin is used as a loading control. (E) Graph showing the percentage of hanging drops containing cells of each condition (WT and GT with and without LIF) that formed EBs. (F) Day 2 EBs for wild-type cells cultured in the presence of LIF, and genetrap AD0857 cells cultured in the presence or absence of LIF (x10). (G) RT-PCR of markers for early embryonic cell fates on day 7 suspension culture EBs. The following markers were used: OCT4 (stem/epiblast), FGF5 (epiblast), SOX2 (stem/epiblast/neural precursor), PAX6 (neural), RAX (anterior neural), NKX2.5 (cardiac mesoderm), FLK1 (endothelial mesoderm), SCL1 (blood), AFP (endoderm), ß-actin (loading control). No RT control is shown for ß-actin.





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