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. 2016 Sep 22;167(1):219–232.e14. doi: 10.1016/j.cell.2016.09.006

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© 2016 The Author(s)

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Figure S1 — Generation of the AAVS1^GFP/TetO7 Reporter Cell Line and Stable Silencing by Targeted DNA Methylation, Related to Figure 1

(A) Schematic of the targeting strategy used to insert the eGFP-expression cassette containing a downstream TetO7 sequence within intron 1 of the PPP1R12C gene (aka. AAVS1). HL: homology arm left; HR: homology arm right; HDR: Homology Driven Repair; ZFNs: Zinc Finger Nucleases.

(B) Gating strategy used to enrich for cells carrying homozygous insertion of the eGFP cassette into AAVS1. Left: flow cytometry dot plot showing K-562 cells at day 30 post-transfection with plasmids expressing the AAVS1-ZFNs and containing the donor sequence. Right: a representative flow cytometry dot plot of the sorted eGFP^bright cells used to derive single-cell clones.

(C) Histogram showing the Mean Fluorescence Intensity (MFI) levels of the indicated clonal populations derived from the sorted cells. In green are highlighted the clones selected for further molecular characterization of the integration.

(D) Southern blot analysis of the indicated populations performed to identify clones containing homozygous insertion of the eGFP-cassette into AAVS1. The red arrows on top of the blot indicate clones selected for subsequent silencing experiments, as they lack signal from the wild-type AAVS1 allele while they contain Targeted Integration (TI) of the cassette. The expected molecular forms of the AAVS1 allele – either wild-type or containing the single cassette or its concatamers – are indicated on the right of the blot.

(E) Schematics of the Bidirectional Lentiviral Vectors (Bid.LVs) expressing tetR:K and mOrange (top) or tetR:DNMT3A and ΔLNGFR (bottom). Ψ: LV packaging signal; SD: Splicing Donor; SA: Splicing Acceptor; mP: minimal Promoter.

(F) Gating strategy used to measure the efficiency of gene silencing within the Bid.LV-transduced cells. An eGFP-negative cell clone, transduced or not with the Bid.LV, was used to set the gate for Bid.LV transduction and eGFP expression.

(G) Representative dot plots of AAVS1^GFP/TetO7 K-562 cells transduced with the indicated Bid.LVs and cultured in the presence of doxycycline for 200 days.

(H) Time-course flow cytometry analysis of 36 independent cell clones derived from the AAVS1^GFP/TetO7 K-562 cells transduced with the Bid.LV-tetR:D3A. Cells were grown for 14 weeks with doxycycline. At 11 weeks post-cloning, the populations were treated for 4 days with 5-aza, and then analyzed for eGFP reactivation by flow cytometry.

(I) Representative flow cytometry dot plots of cells silenced with the Bid.LV-tetR:D3A and treated or not for 7 days with 5-aza.

(J) Gene expression profile of the AAVS1 locus from eGFP-negative cells transduced with the indicated Bid.LVs. The expression level of each gene was normalized to HPRT1 and represented as fold change over matched, untransduced AAVS1^GFP/TetO7 K-562 cell clone (mean ± SEM for Bid.LV-tetR:D3A, n = 3 independent analyses; mean value for Bid.LV-tetR:K, n = 2 independent analyses).