Skip to main content
. 2013 Oct 23;42(2):1365–1378. doi: 10.1093/nar/gkt947

Figure 5.

Figure 5.

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).