At both 37 and 30 ��C, the initial transfection had no significant effect on HBsAg concentration in the culture supernatants. However, the second and third transfections had increasingly inhibitory effects on HBsAg secretion when cells were cultured under hypothermic conditions. As expected, Seliciclib FDA transfection of the C TALEN, which targets a different part of the HBV genome, did not affect HBsAg secretion (Figure 2c,dd). The P1 TALEN and P2 TALEN had modest or no inhibitory effect on HBsAg secretion from HepG2.2.15 cells respectively (Supplementary Figure 6, online). Figure 2 Expression and anti-HBV efficacy of TALENs in cultured cells. (a) Immunodetection of each of the L or R subunits of the S or C TALENs following plasmid transfection of cultured Huh7 cells. Representative fields under 400�� magnification are shown.

… TALEN-mediated targeted mutagenesis of cccDNA To assess targeted TALEN-mediated mutagenesis of cccDNA, circular duplex DNA that had been subjected to ATP-dependent DNase treatment was isolated from HepG2.2.15 cells. PCR-based analyses were initially carried out to assess contamination of the cccDNA sample with cellular genomic DNA and HBV rcDNA (Figure 3a,bb). To detect genomic DNA contamination, primer sets that amplify HBV C DNA or control cellular genomic sequences located in the A1AT gene were used. With the A1AT gene primers, DNA was efficiently amplified from cellular genomic DNA but not when using the cccDNA preparation as template (Figure 3a). Primers targeting the C sequence of HBV amplified DNA from both cccDNA and HepG2.2.15 cell genomic DNA preparations.

Efficiency of amplification was however considerably higher when using the cccDNA preparation as template. Amplification of HBV C sequences from the genomic template was expected, and is likely to be derived from stable HBV integrants within HepG2.2.15 cells. To verify removal of rcDNA, DNA prepared from serum of HBV transgenic mice21 using different procedures was subjected to amplification with S gene primers (Figure 3b). Serum from these mice does not contain cccDNA. Each extracted serum sample contained approximately 5��106 copies of HBV rcDNA, which is a threefold excess of the estimated number of rcDNA copies in the starting material derived from the HepG2.2.15 cells. An S gene amplicon was not detectable when using the serum-derived sample that was subjected to the method used for cccDNA preparation from HepG2.

2.15 cells. This procedure included cccDNA-enrichment and ATP-dependent DNase treatment. Collectively these data indicate that the cccDNA preparation used for the T7 endonuclease assays were not significantly contaminated with either cellular genomic DNA or HBV Brefeldin_A rcDNA. Figure 3 Targeted disruption of cccDNA extracted from HepG2.2.15 cells. (a) PCR analysis using A1AT or HBV C gene primer sets carried out on total genomic DNA or cccDNA isolated from HepG2.2.15 cells.