Figure 5
From: TALEN mediated gene editing in a mouse model of Fanconi anemia
Gene-targeting in FA-A murine HSPCs. (a) Analysis of viability (percentage of DAPI− cells) in FA-A Lin− BM cells nucleofected with the TALEN and the therapeutic PGK-hFANCA donor at 48 hours post-nucleofection. U: untransfected cells; Double negative (-): Nucleofected without DNA; T: 2.5 µg of each NN-TALEN monomer; D: 2 or 4 µg of the therapeutic PGK-hFANCA donor. Data are the mean ± S.D. (n = 7 experiments). (***)P-value <0.001 indicates significant differences with a one-way ANOVA followed by a post-hoc Tukey test. (b) Clonogenic assays of nucleofected Lin− cells under the conditions shown in A). Data are the mean ± S.D. (n = 7 experiments). (*)P-value < 0.05 indicate significant differences with a one-way ANOVA followed by a post-hoc Tukey test. (c) Phenotypic correction measured with clonogenic assays in nucleofected FA-A Lin− BM cells treated with 30 nM MMC compared to cells cultured in the absence of MMC and compared with historical data of resistance to MMC in WT mice. MMC survivals are indicated considering the number of hematopoietic colonies generated without drug selection as 100%. Cells were subjected to nucleofection with 2.5 µg of each TALEN monomer and the therapeutic PGK-hFANCA donor (2 and 4 µg). Data are presented as mean ± S.D. (n = 4–7 with the except of D2 µg where n = 2 experiments). No statistical differences were found among groups with a non-parametric Kruskal-Wallis and median test. d) Representative PCR analysis for the study of the 3′ integration junction (1,337 bp). C+: sequenced genomic DNA positive for the integration junction in edited FA-A MEFs; T + D: samples from nucleofected FA Lin− BM cells with 2.5 μg of each TALEN monomer together with 4 μg of the therapeutic PGK-hFANCA donor; D: 4 μg of the therapeutic PGK-hFANCA donor; IX and λ BstII: DNA molecular weight markers. Analysed colonies are numbered and the positive ones framed in blue.
