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. 2012 Dec 26;41(5):e63. doi: 10.1093/nar/gks1446

Figure 2.

Figure 2.

Structural analysis of TALEN-encoding adenoviral vector genomes. (A) DNA structures of the first-generation adenoviral vectors Ad.ΔE1.TALEN-LS1.F50 and Ad.ΔE1.TALEN-RS1.F50 and of the second-generation adenoviral vectors Ad.ΔE1ΔE2A.TALEN-LS1.F50 and Ad.ΔE1ΔE2A.TALEN-RS1.F50 drawn in relation to those of their respective plasmid clones. The adenoviral vector and shuttle plasmid DNA templates are depicted as BcuI physical maps with numerals specifying the different restriction fragment sizes in kilobases (kb). These DNA molecules harbor a bi-cistronic expression unit coding for the AAVS1-specific artificial nucleases TALEN-LS1 or TALEN-RS1 and, through an IRES sequence, an RFP reporter. The ORFs are under the transcriptional control of the cytomegalovirus immediate-early gene promoter (CMV) and the simian virus 40 (SV40) polyadenylation signal. The central regions of TALEN-LS1 and TALEN-RS1 encode a tandem of 17.5 TALE repeats that define specificity to the AAVS1 target sequence at the human chromosome 19 (19q13.42-qter). C-terminally both TALENs contain the non-specific cleavage domain of FokI, whose catalytic activity is contingent on DNA binding-dependent dimerization. Each of the TALEN proteins are tagged by an HA antigen located close to their N-termini (not drawn). The thick and thin lines represent adenoviral vector and plasmid backbone sequences, respectively. Ψ, human adenovirus serotype 5 packaging signal; Ad L-ITR and Ad R-ITR, ‘left’ and ‘right’, respectively, adenoviral inverted terminal repeat. F50, ORF encoding chimeric fiber composed of basal shaft domains from adenovirus serotype 5 fused to apical shaft and knob motifs from adenovirus serotype 50. The kanamycin-resistance gene (KanR) and the prokaryotic origin of replication (ori) are also indicated. (B) Restriction fragment length analysis of Ad.ΔE1.TALEN-LS1.F50 and Ad.ΔE1.TALEN-RS1.F50 genomes treated with BcuI. Plasmids pAd.ΔE1.TALEN-LS1.F50 and pAd.ΔE1.TALEN-RS1.F50 served as a reference. The sizes (in kb) of the DNA restriction fragments resulting from the BcuI treatment are indicated on the left. The arrowhead points to the 2.4-kb DNA fragments diagnostic for intact, non-rearranged, TALE-derived DNA sequences within TALEN-encoding genes. Marker, molecular-weight marker Gene Ruler DNA Ladder Mix. (C) PCR analysis on DNA isolated from Ad.ΔE1.TALEN-LS1.F50 and Ad.ΔE1.TALEN-RS1.F50 particles using primers TALEN-Seq-F and TALEN-Seq-R (F and R half arrows, respectively). These primers, drawn in relation to their respective target sequences, amplify most of the TALEN-LS1 and TALEN-RS1 DNA including the 17 102-bp tandem DNA repeats. The position (arrowheads) and size (in kb) of the 2.1-kb amplicon diagnostic for intact TALEN transgenes are indicated at the right. Marker, Gene Ruler DNA Ladder Mix. H20, PCR performed with nuclease-free water instead of vector DNA template. (D) Autoradiogram of MunI-treated extrachromosomal DNA isolated from HeLa cells transduced with Ad.ΔE1.TALEN-LS1.F50 or with Ad.ΔE1.TALEN-RS1.F50 at MOIs of 10, 5 and 1 HTU/cell. The DNA, extracted at 3 days post-transduction, was subjected to Southern blot analysis using a 397-bp probe hybridizing to a sequence encoding the NH2 terminus of the TALEN-LS1 and TALEN-RS1 proteins.