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. 2020 Dec 20;296:100189. doi: 10.1074/jbc.RA120.015152

Figure 3.

Figure 3

Manipulation of the −5 CRE.A, the −5 CRE was flipped by inserting opposing loxP sites followed by tamoxifen (4OHT) treatment. Left panel, schematic; right panel, mRNA levels of Nanog and relevant pluripotency genes in individual isolated clones. n = 3. B, an approximately 2-kb region between the −5 CRE and Nanog TSS was deleted. Left panel, schematic; right panel, Nanog mRNA levels. n = 5. C, two copies of the −5 CRE were inserted downstream of Nanog in cells where the endogenous enhancer is floxed. Endogenous enhancers were deleted via treatment with tamoxifen. Left panel, schematic; right panel, mRNA levels of Nanog in bulk cells treated with vehicle or 4OHT. n = 3. All mRNA levels measured by RT-qPCR and shown as 2∧ΔΔCT compared with wildtype. ∗p < 0.05, ∗∗p < 0.01 Student’s two sample t test. None of the cell lines shown express Nanog in trans. CRE, cis-regulatory element.