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. 2022 Mar 17;8(3):245–256. doi: 10.1038/s41477-022-01108-y

Fig. 1. Generation of transgenic tobacco lines expressing TALENs targeted to the mitochondrial nad9 locus.

Fig. 1

a, Binding sites of the three designed TALEN pairs in the nad9 coding sequence (CDS) and schematic view of the binary vectors used for plant transformation. The nucleotide positions refer to the start codon (ATG = 1). The scissors indicate the predicted TALEN cut sites; the BsrGI restriction site is boxed. LB/RB, T-DNA left/right borders; 35S, CaMV 35S promoter; mt PS, mitochondrial presequence (from the Arabidopsis IVD protein); HA, 3xHA-tag; TALE N term, TALE amino terminus; Repeats, TALE DNA-binding units; FokI, endonuclease domain; OCS, octopine synthase terminator (Agrobacterium); FLAG, 3xFLAG-tag; NOS (octagon), nopaline synthase terminator (Agrobacterium); nptII (kanR), neomycin phosphotransferase II encoding kanamycin resistance; NOS (arrow), nopaline synthase promoter (Agrobacterium). The coloured arrows indicate TALEN binding sites. b, Detection of TALEN activity by Southern blot analysis. The predicted sizes of hybridizing restriction fragments and the location of the probe are depicted. The scissors mark the cut sites of the restriction enzymes PvuII, EcoRV and BsrGI, and of the pJF1006-encoded TALEN pair. BsrGI cuts 2 bp away from the predicted TALEN cut site. In addition to PvuII and EcoRV, BsrGI was included in a digestion reaction with wild-type DNA (WT + BsrGI; right) to simulate the TALEN cut. The numbers below the gel give the relative intensities of the TALEN cleavage products. The arrowheads mark the positions of the three expected signals (magenta indicates uncut; cyan indicates TALEN-cut). c, PCR and RT–PCR assays to test for the presence of TALEN arms in the lines shown in b. The binding sites of the PCR primers are shown in the upper panel (the dashed lines are for RT–PCR primers), and the lower panels display the PCR results. A sequence from the β-TUBULIN gene was amplified as an internal control. The arrowheads indicate the expected sizes for β-TUBULIN (black, 412 bp/303 bp for DNA/cDNA), the HA arm (cyan, 311 bp/210 bp) and the FLAG arm (magenta, 251 bp/212 bp). H2O, water control; M, DNA size marker. Only the FLAG–TALEN arm is present in Nt-JF1006-30. Pools of kanamycin-resistant T1 seedlings obtained by selfing were used for the nucleic acid extractions. See also Extended Data Fig. 4. The experiments in b and c were performed once.