Figure 1
From: TALEN mediated gene editing in a mouse model of Fanconi anemia
Mbs85-specific TALEN and donor-mediated targeted insertion in FA MEFs. (a) 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 therapeutic hFANCA 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; 2A: 2A self-cleaving peptide sequence; SV40pA: simian virus 40 polyA sequence; PuroR: Puromycin resistance gene. (b) Flow chart indicating the study design and the different analyses performed in gene-edited cells of FA-A MEFs. (c) Analysis of viability (percentage of DAPI− cells) in the different conditions. U: untransfected cells; T: 2.5 µg of each TALEN monomer; D: donor doses (0.75, 2 or 4 µg). Bars indicate the mean ± S.D. (n = 2 experiments). d) Cleavage efficacy of the Mbs85-specific TALENs analysed by Surveyor assay. Representative electrophoresis gel showing the disruption of the target locus in FA-A MEFs nucleofected with only the TALENs (T, 2.5 µg of each TALEN monomer) or together with different donor doses (0.75 µg and 4 µg). U: untransfected cells. Samples not digested with the Cel1 endonuclease were used as controls. The extent of TALEN cleavage, measured as the mean percentage of modified alleles, is indicated below. Arrows indicate the size of the parental band (405 bp) and the expected positions of the digestion products (224 bp and 181 bp), that are also indicated with asterisks. IX: DNA molecular weight marker. e) Schematic representation of the targeted integration of the therapeutic PGK-hFANCA donor into the Mbs85 locus of FA-A MEFs. Arrows represent the primers, forward (Fw) and reverse (Rv) used to evaluate the site-specific integration and the size of the PCR amplicon is indicated for each integration junctions. The electrophoresis gel below is a representative image of the integration analysis performed on the same samples as indicated in (c). W: water control; IX: DNA molecular weight marker.
