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1.
Figure 2.

Figure 2. From: Nuclease-mediated gene editing by homologous recombination of the human globin locus.

High-frequency gene targeting using β-globin TALENs. (A) Schematic of nuclease-mediated gene targeting to the endogenous β-globin locus. (B) β-Ubc-GFP targeting vector. Ubc, ubiquitin C promoter; pA, BGH polyadenylation signal sequence. (C) Gene targeting of β-Ubc-GFP to the endogenous β-globin locus in K562 cells using βL4/βR4 TALENs and ZFNs (*P < 0.005 compared to targeting vector alone). Each experimental condition was performed in biological triplicate.

Richard A. Voit, et al. Nucleic Acids Res. 2014 January;42(2):1365-1378.
2.
Figure 1.

Figure 1. From: Nuclease-mediated gene editing by homologous recombination of the human globin locus.

TALEN-mediated disruption at the human β-globin and γ-globin loci. (A) Schematic of the βL4/βR4 TALEN binding site in the human β-globin gene. The ATG start codon and the sickle mutation are highlighted. (B) β-globin gene disruption in HEK293T cells. Arrows indicate specific Surveyor nuclease cleavage products (C) SMRT sequencing of β-globin alleles mutated by treatment with βL4/βR4 TALENs. The 11 most abundant mutated alleles are shown, and the frequency of each is indicated. TALEN binding sites are underlined. (Δ represents deletions, + represents insertions). (D) Schematic of the γL3/γR2 TALEN binding site in the human γ-globin gene. The ATG start codon is highlighted. (E) γ-globin gene disruption in K562 cells. (*, non-specific cleavage product). (F) SMRT sequencing of γ-globin alleles mutated by treatment with γL3/γR2 TALENs. The 11 most abundant mutated alleles are shown, and the frequency of each is indicated. TALEN binding sites are underlined.

Richard A. Voit, et al. Nucleic Acids Res. 2014 January;42(2):1365-1378.
3.
Figure 3.

Figure 3. From: Nuclease-mediated gene editing by homologous recombination of the human globin locus.

Targeting therapeutic β-globin cDNA to the endogenous β-globin locus. (A) β-in-frame-cDNA targeting vector. When targeted, the cDNA is expressed from the endogenous ATG start site. pA, BGH polyadenylation signal sequence; Ubc, Ubiquitin C promoter; MGMT, Methylguanine methyltransferase. (B) Schematic of the wt (top) and targeted (bottom) β-globin locus. The absence of intron 1 in the cDNA-targeted locus results in a shorter PCR product. (C) Results from SMRT sequencing of targeted β-globin alleles without selection (top row) and with up to three rounds of drug selection.

Richard A. Voit, et al. Nucleic Acids Res. 2014 January;42(2):1365-1378.
4.
Figure 4.

Figure 4. From: Nuclease-mediated gene editing by homologous recombination of the human globin locus.

Generation of β-globin reporter cells by in-frame GFP targeting to the endogenous β-globin locus. (A) β-in-frame-GFP targeting vector. When targeted, GFP is expressed from the endogenous ATG start site. Arrows indicate PCR primers used in Panel D to confirm targeting. pA, BGH polyadenylation signal sequence; Ubc, Ubiquitin C promoter; MGMT, Methylguanine methyltransferase. (B) Targeting of β-in-frame-GFP to the endogenous β-globin locus using βL4/βR4 TALENs and ZFNs (*P < 0.05 compared to targeting vector alone). Each experimental condition was performed in biological triplicate. (C) FACS plots of targeted clones, classified by level of GFP expression. (D) Genomic PCR using primers in Panel A to detect presence of a targeted β-globin locus in 11 of 12 GFP positive clones. n.t., no template control; wt, genomic DNA from wild-type cells; pop, genomic PCR from targeted population; –, no sample.

Richard A. Voit, et al. Nucleic Acids Res. 2014 January;42(2):1365-1378.
5.
Figure 6.

Figure 6. From: Nuclease-mediated gene editing by homologous recombination of the human globin locus.

Using fluorescent reporter cell lines to screen globin-modulating compounds. (A) Effect of 400 µM hydroxyurea on β- and γ-globin transcript levels (gray bars) and on GFP and tdTomato expression in targeted cell lines (white bars). Effect of drug treatment on (B) tdTomato expression in γ-globin tdTomato cells, (C) GFP expression in β-globin-GFP cells and (D) the ratio of tdTomato/GFP expression in γ-globin-tdTomato and β-globin-GFP cells. FACS plots showing the effect on the expression of tdTomato and GFP from the γ-globin and β-globin loci of (E) guanosine, and (F) apicidin. Drug concentrations: 0.2% DMSO, 10 µM pomalidomide, 400 µM GDP, 5 µM hemin, 400 µM cGMP, 200 µM GTP, 200 nM mithramycin, 200 µM zileuton, 4 mM phenylacetate, 400 µM GMP, 10 µM decitabine, 1200 µM sodium butyrate, 5 µM cytarabine, 4 µM cisplatin, 400 µM hydroxyurea, 400 nM apicidin, 400 µM guanosine and 400 µM guanine.

Richard A. Voit, et al. Nucleic Acids Res. 2014 January;42(2):1365-1378.
6.
Figure 5.

Figure 5. From: Nuclease-mediated gene editing by homologous recombination of the human globin locus.

Generation of fluorescent γ-globin and dual-globin reporter cell lines. (A) γ-in-frame-tdTomato targeting vector. When targeted, tdTomato is expressed from the endogenous ATG start site. Arrows indicate PCR primers used in Panel E to confirm targeting. pA, BGH polyadenylation signal sequence; PGK, Phosphoglycerate kinase promoter; neoR, Neomycin phosphotransferase. (B) Targeting of γ-in-frame-tdTomato to the endogenous γ-globin locus using γL3/γR2 TALENs (*P < 0.005 compared to targeting vector alone). Each experimental condition was performed in biological triplicate. (C) FACS plots showing stable integration of tdTomato on day 20 in the presence of TALENs. (D) Schematic of the targeted globin loci showing tdTomato being expressed from the endogenous γ-globin ATG start site and GFP from the endogenous β-globin ATG start site. The wild-type γ- and β-globin gene sequences are still present after targeting but are not expressed because of the stop codons that follow the targeted tdTomato and GFP sequences. (LCR, locus control region). (E) Genomic PCR using primers in Panel A to detect presence of a targeted γ-globin locus in samples treated with γ-globin TALENs (Pair 1: γL3/γR2, pair 2: γL3/γR3, pair 3: γL2/γR2). Wild-type cells (left) were targeted to generate the γ-globin tdTomato reporter line, and β-globin-GFP cells (right) were targeted to generate the dual reporter cell line. (F) FACS plots showing the fluorescent profile of the β-globin-GFP reporter (left) the γ-globin-tdTomato reporter (center) and β-globin-GFP/γ-globin-tdTomato dual reporter (right).

Richard A. Voit, et al. Nucleic Acids Res. 2014 January;42(2):1365-1378.

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