Fig. 3. 5T4 is transcriptionally upregulated on ES cells following removal of LIF and the antigen associates with all primary germ layers. (a) 5T4 transcript expression in differentiating ES cells. (i) MESC and (ii) OKO160 ES cell lines were differentiated for 12 days as monolayer cultures by removal of LIF from the growth medium. cDNA was prepared at the specified time points, as described in the legend to Fig. 2, and semi-quantitative RT-PCR analysis (25 cycles) of m5T4 was performed. Samples were run on 2% agarose gels containing ethidium bromide and visualised on a UV transilluminator. ß-tubulin (ß-tub; housekeeping gene) is included for standardisation. D0, undifferentiated cells; D12, 12 days following removal of LIF. (b) 5T4 antigen is expressed on cells derived from all three germ layers following removal of LIF from ES cells. MESC ES cells were differentiated for 3, 6 and 9 days as monolayer cultures by removal of LIF from the growth medium and 5T4-positive cells purified using anti-5T4 monoclonal antibody 9A7 and MidiMACS LS columns. cDNA was prepared as described above followed by RT-PCR analysis of various germ layer-specific transcripts (see Table 1). Samples were run on 2% agarose gels containing ethidium bromide and visualised on a UV transilluminator. ß-tub (housekeeping gene) is included for standardisation. D3, 3 days following removal of LIF; D9, 9 days following removal of LIF.

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