Fig. 2. Expression of cloned PCR products in pluripotent cells. (A) Northern blot analysis of 4 µg poly-A+ RNA, isolated from ES cells and EPL cells grown for 2, 4 or 6 days in 50% MEDII in the presence (+) or absence (–) of LIF, probed for Oct4, CRTR1, Rex1, Psc1, Fgf5 and mGAP. Transcript sizes were: Oct4, 1.6 kb; CRTR-1, 9.4 kb; Rex1, 1.9 kb; Psc1, 5.5 kb; Fgf5, 2.7 kb; and mGAP, 1.5 kb. (Bi) Northern blot analysis of 25 µg total RNA isolated from ES cells and EPL cells grown for 2, 4, 6 or 8 days in 50% MEDII in the presence of LIF (+) and probed for PRCE and mGAP expression. Transcript sizes were: PRCE, 6.6 kb; mGAP, 1.5 kb. (Bii) Quantitation of northern in (Bi). PRCE expression was normalized against the mGAP loading control and expressed as a ratio, with ES cells assigned a value of 1. (C) In situ hybridization of ES cells (i, iii) and EPL cells cultured for 2 days in the presence of MEDII + LIF (ii, iv) with Oct4 (i, ii) and PRCE (iii,iv) antisense digoxygenin-labelled riboprobes. Images were captured using Hoffmann interference contrast microscopy. Bar, 90 µm. (D) In situ hybridization of EPL cells cultured for 4 days in the presence of MEDII and LIF with CRTR-1 (i, ii) and PRCE (iii, iv) antisense digoxygenin-labelled riboprobes. (i, iii) Phase contrast microscopy; (ii, iv) bright field microscopy. Arrows denote differentiated cells. Bar, 25 µm. Equivalent cell populations to those illustrated in C and D were probed with digoxygenin-labelled sense probes and failed to develop colour (data not shown).
