Figure 3.
CRISPRa Gene Induction and cDNA-Mediated Overexpression of Sall1, Nanog Reprogrammed MEF to iPSCs
(A) (4F + CRISPRa) MEFs stably transfected with CAG4F and gRNAs against Sall1/Nanog/Sall1+Nanog in ESC medium (Oct4-GFP+ve colonies after 18 days; mean of 3 independent experiments ± SD, ∗∗p < 0.01; ∗∗∗p < 0.001) (4F + cDNA) MEFs stably transfected with TRE4F, TRENanog, and TRESall1 (all co-transfected with PBEF-1αTet3G), induced with 0.5 μg/mL Dox for 12 days and counted on day 18 (mean of 3 independent experiments ± SD, ∗∗p < 0.01).
(B) Alkaline phosphatase-positive (AP+ve)-stained ESC colonies reprogrammed from MEFs by 4F alone and in combination with Sall1 (induced with Dox at 0.5 μg/mL).
(C) iPSCs reprogrammed from C57B/6J MEF with 4F/Sall1. Oct4-GFP expression and ESC-like morphology (upper panel), immunofluorescent staining for pluripotency markers SSEA-1 and NANOG (lower panel).
(D) In vitro differentiation of C57B/6 MEF reprogrammed iPSCs with 4F/Sall1; neuronal differentiation in N2B27 (immunofluorescence for β-tubulin III+); mesoderm and endoderm differentiation in M10 (alpha smooth muscle actin [α-SMA] and alpha fetoprotein [AFP] antibody staining).
(E) Chimeric mice produced with 4F/Sall1-iPSCs injected into CD1 blastocysts.
(F) Inactivation of X chromosomes in female 4F/Sall1-iPSCs (co-immunostaining for H3K27me3 and Oct4. Arrows indicate H3K27me3 foci).