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. 2015 Dec 1;29(23):2420–2434. doi: 10.1101/gad.271783.115

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© 2015 Pech et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 1. — A Tert promoter knock-in reporter accurately reflects telomerase activity in both pluripotent and differentiating ES cells. (A) Generation of a knock-in transcriptional Tert reporter. TdTomato was inserted at the initiating methionine of Tert, with a floxed resistance cassette placed into the first intron. (B) Phase contrast and epifluorescent microscopy of Tert-Tomato reporter in Tert^Tomato/+ mES cells grown in LIF/2i conditions. Bar, 200 μm. (C) Expression of Tert-Tomato reporter in Tert^Tomato/+mES cells grown in LIF/2i, quantified by FACS. Untargeted parental mES cells are shown in black. Results were consistent across independently targeted clones. (D) Expression of fluorescent reporter genes by FACS in doubly targeted Tert^Tomato/+ Oct4^ires-EGFP/+ mES cells grown in LIF/2i conditions. Gates were drawn relative to parental and singly targeted mES cells. (E) Schema for in vitro differentiation of mES cells to an adipogenic fate. (EB) Embryoid body; (RA) retinoic acid; (I,T,R) insulin, triiodothyronine, and rosiglitazone. (F) Oil-Red-O staining of mES cells at day 20 of differentiation. Bar, 500 μm. (G) Fluorescent reporter expression by FACS in Tert^Tomato/+ mES cells during adipogenic differentiation. Gates were drawn based on the background fluorescence of untargeted parental mES cells. The percent of events in each gate is shown. (SSC) Side scatter. (H) Telomerase activity measured by telomere repeat amplification protocol (TRAP) in populations shown in G. CHAPS and H₂0 represent buffer-only negative controls. TRAP reactions were programmed with 3000 or 1000 FACS-sorted cell equivalents.