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. 2013 Dec 24;22(2):303–311. doi: 10.1038/mt.2013.212

Figure 5.

Figure 5

TALENs result in a decreased cccDNA level and misrepaired cccDNA. Huh7 cells were co-transfected with the linear full-length HBV DNA and the plasmids encoding indicated TALENs. (a,b) The levels of intracellular cccDNA 48 hours post-transfection were analyzed by PCR (a) and real-time PCR (b) using primers for cccDNA quantitation. (c) The cccDNA was extracted from the control and TALENs-expressing cells as described in methods. The DNA segments encompassing the recognition sites of TALENs-L2/R2 were PCR-amplified, and the DNA amplicons were melted and annealed. The annealed DNA was treated with five units of the mismatch-sensitive T7 endonuclease 1 (T7E1) for 15 minutes at 37 °C and then assessed by agarose gel electrophoresis. (d) Huh7 cells were co-transfected with the linear full-length HBV DNA and indicated plasmids. Forty-eight hours post-transfection, the supernatants were collected to detect HBeAg by ELISA. ELISA, enzyme-linked immunosorbent assay; HBV, hepatitis B virus; TALEN, transcription activator-like effector nucleases.