(A) Diagram showing the chemical-genetic strategy to selectively inhibit
GSK3α or GSK3β.
(B) Phase-contrast images of
Gsk3β−/− +
Gsk3β-L132G and
Gsk3α−/− +
Gsk3α-L195G ESCs plated at a clonal density and
cultured in N2B27 medium supplemented with the indicated inhibitors for 7 days.
Representative images show results from thee independent clones of mutant ESC
lines with GSK3 transgene levels similar to the physiological levels. Scale bar,
100 μm.
(C) Quantification of AP+ colonies of different ESC lines. ESCs were
plated onto six-well plates at a density of 1,000 cells/well and cultured for 7
days in N2B27 medium supplemented with the indicated inhibitors. Data represent
means ± SD of three biological replicates. **p <
0.01.
(D) Strategy and sequencing result for introducing the L132G point mutation at
the Gsk3β locus in mouse ESCs by CRISPR/Cas9. The
single guide RNA (sgRNA) target site is shaded in gray. A targeting vector
containing 5′ homologous arm-loxP-CAG-PuroR-loxP-3′ homologous
arm was used as the template for homologous recombination. The 5′-NGG
PAM site (TGG) is underlined in the wild-type locus. The mutated code
(CTG→GGT) is highlighted in red. The restriction fragment length
polymorphism (RFLP) site is labeled in red and indicated by an asterisk. This
RFLP site (AˆCGT) can be digested by HpyCH4IV. Sequencing result showing
the knockin of the CTG→GGT (underlined in red) mutation at the
Gsk3β-L132G locus in one of the ESC clones.
(E) Representative images of wild-type and Gsk3β-L132G
knockin (Gsk3β-L132G-KI) ESCs cultured in N2B27 medium
supplemented with PD03 and 3MB-PP1 for 5 days.
Gsk3β-L132G-KI ESCs self-renewed continuously in
PD03 + 3MB-PP1, whereas wild-type ESCs differentiated rapidly under the
same conditions. Scale bars, 100 μm.