All mice were maintained on NTBC throughout. Livers were harvested 2 weeks after treatment. For stable integration studies, d3 Fah5981SB neonates were injected with AAV2-Fah at 1 × 1011 vg in 10 μL volume by intravenous facial vein injection. Littermate controls were similarly injected with isotonic NaCl solution. Mice were maintained on NTBC until weaning and then withdrawn to select for corrected hepatocytes. Eleven weeks after treatment, a two-thirds partial hepatectomy

was performed to induce liver regeneration.32 Livers were harvested >12 weeks after surgery. For random integration studies, d3 Fah5981SB neonates were coinjected with 4 × 1010 vg of both AAV8-Fah and AAV8-hAAT (in 10 μL volume) by intravenous CP-868596 price facial vein injection. Mice were maintained on NTBC until weaning and then withdrawn to select for corrected hepatocytes. Serum (for liver function tests) and liver tissue were collected at harvest. Adult Fah5981SB mice (age 8-12 selleck inhibitor weeks) were injected

with 1 × 1011 vg of AAV8-Fah (in 100 μL volume) by intravenous tail vein injection. Age-matched littermate controls were similarly injected with isotonic NaCl solution. Mice were placed on NTBC as needed. Serum and liver tissue were harvested >12 weeks after treatment. In both adult and neonatal experiments, a minimum of two liver sections were analyzed per mouse and evaluated for the number of FAH+ cell clusters, each representing the clonal expansion of a single corrected hepatocyte. Clonal frequencies, correction factors, hepatocyte counts, fixation, and immunohistochemistry

protocols were done as described.33 Quantitation was performed by two separate, blinded investigators. Experimental results were analyzed for significance by applying a student 2-tailed t-test assuming equal variance. P values <0.05 were considered statistically significant. AAV vector preparation and titering were performed according to standard AAV protocols as described.34 For serial transplantation surgeries, livers were isolated from corrected mice and 3 × 105 to 5 × 105 random hepatocytes were injected intrasplenically at 100 μL volume into Fah5981SB recipient mice as described.35 Total RNA was isolated from randomly dissected liver tissue with an RNeasy Mini kit (Qiagen). The cDNA was produced MCE with a Superscript III First-Strand Synthesis kit (Invitrogen). PCR was performed on an iCycler (Bio-Rad Laboratories). Reverse transcription (RT) reaction (100 ng) was subjected to two-step PCR amplification under the following conditions: 1 cycle 95°C × 3 minutes, followed by 45 cycles of 95°C × 15 seconds and 68°C × 50 seconds. Primer sequences: Fah forward: 5′-AGAACTTACTGTCTGCCAGCCAAG-3′; Fah reverse: 5′-GAGGACCATCCCGAAAATGTG-3′; glyceraldehyde 3-phosphate dehydrogenase (Gapdh) forward: 5′-CCACCCCAGCAAGGACACTG-3′; Gapdh reverse 5′-GCTCCCTAGGCCCCTCCTGT-3′. All samples were subjected to ± RT controls and results were normalized to Gapdh expression.