Figure 1: Constitutive expression of a Zscan4c–ERT2 fusion protein increases developmental potency.
From: Zscan4 restores the developmental potency of embryonic stem cells
(a) The structure of a Zscan4c–ERT2 fusion protein. Zscan4c contains one SCAN domain and four C2H2 zinc finger domains. (b) Fluorescence microscopy of MC1-ZE3 cells, in which a Zscan4 promoter drives the expression of Emerald marker (left), MC1-ZE3-ZERT2 clone #15 cells, in which the Zscan4c–ERT2 fusion protein is constitutively expressed, cultured in the absence of Tmx (middle), MC1-ZE3-ZERT2 clone #15 cells cultured in the presence of Tmx for 3 days (right). Scale bar, 50 μm. (c) Flow cytometry analysis of MC1–ZE3 ES cells (left, control) and MC1–ZE3–ZERT2 #15 ES cells (right) in the absence or presence of 1 μM Tmx. Em fluorescence levels (average±s.e.m.; n=6) are shown. Note threefold increase of Em+ cells by the constitutive expression of a Zscan4c–ERT2 fusion protein even without Tmx. (d) Quantitative RT–PCR analysis of endogenous Zscan4 expression measured by using PCR primer pairs specific for 3′ UTR of Zscan4 in MC1–ZE3 ES cells (left, control) and MC1–ZE3–ZERT2 #15 ES cells (right) in the absence or presence of 1 μM Tmx. The fold induction of endogenous Zscan4 expression levels (average±s.e.m.; n=6) compared with that of control MC1–ZE3 is shown. Note the sixfold increase of endogenous Zscan4 at the RNA level by the constitutive expression of a Zscan4c–ERT2 fusion protein even without Tmx. (e) V6.5 parental ES cells (passage number 14), V6.5 ZERT2 #2 (p.20), V6.5 ZERT2 #18 (p.22) ES cell colonies after whole-mount RNA in situ hybridization of a Zscan4 full-length probe (which detects both endogenous and exogenous Zscan4 RNAs: upper panel) or a Zscan4 3’-UTR probe, which detects only endogenous Zscan4 RNAs. Scale bar, 100 μm. (f) Representative coat colours of chimeric mice generated by injecting various ES cells into blastocysts. Higher chimerism represents higher contribution of injected ES cells to mice, indicating higher developmental potency of the ES cells. (g) Percentage distribution of chimerism levels among ‘n’ number of mice born from various ES cell lines.
