Figure 3.

The AAVS1 Locus Mediates Transgene Silencing by DNA Methylation in Undifferentiated hESC

(A) The RMCE donor vectors pZ:F3-P OCT4p-GFP-F (top) and pZ:F3-P (cHS4)X4 OCT4p-GFP-F (bottom) contain the OCT4p-GFP-polyA cassette without or with insulators (blue arrows), respectively.

(B) Representative data of GFP expression levels (microscopy and FACS) in undifferentiated wild-type (WT), master cell line (MCL) and OCT4p without (OCT4p GFP) or with insulators (In OCT4p GFP). Scale bar, 100 μm.

(C) Middle: representation of the AAVS1 locus adapted from Ogata et al. (2003). Thin line: 4-kb-long sequence containing the AAVS1 locus. Empty boxes: DNase hypersensitive region (DHR), Rep-binding site (RBS), and integration site (IS). Exon 1 (black box), homology regions used for homologous recombination (striped boxes), and ZFN targeting site (gray line) are depicted. Bisulfite sequenced regions analyzed in wild-type (WT) undifferentiated hESC: blue, gray, and red thick lines. The OCT4p-GFP RMCE lines (+/− insulators) and the fragment analyzed by bisulfite sequencing (purple line) are below the ZFN site. Top and bottom show representative panels and percentages of the results of bisulfite sequencing of the regions highlighted in the middle figure (n = 2 IEs).

(D) Top, scheme of the hepatic differentiation protocol: Activin (A), W (Wnt3a), B4 (BMP4), aF (aFGF), H (HGF), UD (undifferentiated), DE (definitive endoderm), HE (hepatic endoderm), HB (hepatoblasts), and HP (hepatocytes). Bottom, OCT4 expression levels as percentage of GFP⁺ cells and mRNA expression levels (relative expression [RE], mean ± SEM of n = 3 IEs).

(E) Immunocytochemistry of day 8 differentiated progeny. Scale bar, 100 μm.