Figure 4
From: TALEN mediated gene editing in a mouse model of Fanconi anemia
Gene-targeting in WT murine HSPCs. (a) Flow chart indicating the study design and the different analyses performed in gene-edited murine HSPCs. (b) Schematic showing the architecture of the murine Mbs85 locus, with the target sites of the TALENs highlighted, and the structure of the donor used with the EGFP reporter cassette driven by the phosphoglycerate kinase promoter (PGK) flanked by sequences homologous to the genomic target locus. The resulting locus upon targeted integration (TI) of the donor is indicated in the lowest part of the panel. mHA-L and mHA-R: homology arms to the murine Mbs85 locus; SV40pA: simian virus 40 polyA sequence; (c) Analysis of viability (percentage of DAPI− cells) and percentage of EGFP+ cells in WT Lin− BM cells nucleofected with the TALEN and the PGK-EGFP reporter donor at 48 hours post-nucleofection. U: untransfected cells; Double negative (-): Nucleofected without DNA; T: different doses of TALEN monomers (from 0.75 to 2.5 µg); D: 2 or 4 µg of the PGK-EGFP reporter donor. Data are the mean ± S.D. (n = 5 experiments). (***)P-value < 0.001 indicates significant differences with a one-way ANOVA followed by a post-hoc Tukey test. d) Clonogenic assay to evaluate the ability of WT Lin− BM cells to generate hematopoietic colonies under the conditions shown in C). Data are the mean ± S.D. (n = 5 experiments). (*)P-value < 0.05, (**)P-value < 0.01 indicate significant differences with a one-way ANOVA followed by a post-hoc Tukey test. (e) Targeted integration percentage of the PGK-EGFP reporter donor into the Mbs85 locus of WT Lin− BM cells for the 3′ integration junction calculated in the hematopoietic colonies that were positive for the PCR. Percentages calculated in nucleofected cells with different doses of TALEN (with triangles, 0.75 µg of each monomer; with circles, 2.5 µg of each monomer) and the donor (in white, the dose of 2 µg; in black, the dose of 4 µg). Data are the median ± interquartile range (n = 5–9). (*)P-value < 0.05 indicates significant differences with a Mann-Whitney test.
