Targeting BLG in goat fibroblasts.

(A) Schematic overview depicting the targeting strategy for the BLG locus. Blue boxes, exons of BLG; Diagonal lines above, TALEN1/2 pair binding sites; Level arrow lines, primers used for PCR; The predicted size of southern hybridization bands with BamHI digestion, for both the endogenous BLG locus and the BLG targeted locus, is indicated. (B) PCR-based measurements of TALEN-driven exogenous gene integration into the BLG locus in goat fibroblasts. Cells were right untransfected (lane 4, for negative control) or were transfected with an expression cassette for TALENs that induce a DSB at exon1 of BLG locus (lane 3), and donor plasmids carrying a foreign gene flanked by two homology arms, in the absence (lane 2) and presence (lane 1) of the TALEN. (C) 3’ Junction PCR results from cell lysis templates. TALEN-mediated transgene insertion yielded 2,300-bp PCR products using primers B31 and B32, which were specific to the neo gene and the BLG locus, respectively. (D) 5’ Junction PCR analysis performed on genomic DNA of 3’ junction PCR-positive colonies using primers B51 and B52 to amplify the 1,800-bp left-hand junction between the endogenous BLG locus and the exogenous hLA.