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. 2008 Sep 5;4(9):e1000170. doi: 10.1371/journal.pgen.1000170

Figure 6. Reprogramming is self-sustaining and can be maintained in the absence of ES-derived Oct4.

Figure 6

(A) To address whether reprogramming is stable or subject to reversion, we ablated Oct4 expression after hybrid formation. ZHBTc4 ES cells [mES with endogenous Oct4 replaced by an inducible transgene (Oct4βgeo) which can be downregulated by addition of doxycycline (Dox)] were fused with mouse B-lymphocytes (mB) carrying a GFP transgene under the control of Oct4 promoter (GOF18ΔPE). Reprogramming of mB results in the re-activation of GFP in hybrid colonies (d10, lower panels). Kinetic analysis of single cells (upper panels) showed that transgene re-activation occurs in heterokaryons (day 2, 2 arrows), and hybrid cells (day3, arrowhead). mB cells are shown as negative controls. Nuclei were visualised with DAPI staining (blue). Scale bars, 10 µm. (B) Hybrid clones (mES x mB, 4n) that re-expressed GFP were isolated and analysed by FACS. mES, mB and mES x mB hybrid cells unstained (left panel) or stained with propidium iodide (right panel) to assess GFP expression and DNA content, respectively. (C) Hybrid clones (4 and 12) were treated with Dox to ablate ES-derived Oct4, and quantitative RT-PCR confirmed downregulation of Oct4βgeo transcript (upper panels). Removal of mES-derived Oct4βgeo in hybrid clones did not affect gene expression of pluripotency-associated transcripts (lower panels; mOct4, mNanog and mSox2) after 96 hours of Dox treatment. (D) No differentiation was observed after Oct4 removal in hybrid cells. mRex1 expression was retained and the extra-embryonic markers mHand1 and mCdx2 were not induced. In ZHBTc4 ES cells (open bars) upon Dox treatment, mHand1 and mCdx2 were induced [36] and are shown for comparison. Data were normalised to mGapdh expression. Error bars indicate the s.d. of 3 independent experiments. (E) Western blotting with anti-Oct4 antibody confirmed that Oct4 protein is rapidly removed after Dox treatment of ZHBTc4 ES cells (lower panel) but remains detectable at all times in hybrid cells (upper panels). Equivalent protein loading is shown with Lamin B detection.