3B). Normal tissue stained

uniformly positive for the Fah protein (Fig. 3B, I), whereas it was undetectable in nodular areas (Fig. 3B II-V) except for some displaced tissue surrounding the tumor-like structures (Fig. 3B III). The hypothesis that LV-mediated insertional mutagenesis was not associated with tumor formation was supported by the fact that tumorous tissue had low copy numbers (0.01 ± 0.02) compared to histologically normal areas expressing the Fah protein (0.40 http://www.selleckchem.com/products/Nolvadex.html ± 0.04; P < 0.05) (Fig. 3C; Supporting Fig. 5). Even in the absence of hepatic tumors, LV integration could initiate clonal imbalance by activating growth promoting genes as it was demonstrated in gene therapy of the hematopoietic system.35, 36 To test for this, LV integration sites from serially transplanted hepatocytes of the in vivo (n = 25) and the ex vivo group (n = 13) were amplified by LM-PCR and 454 pyrosequencing. In a total of 296,036 sequences we identified 4,349 independent insertion sites from 38 repopulated animals, which located to 2,483 unique gene IDs (GID). Numerous insertion sites were found in all generations of serially transplanted mice with no dominant bands in agarose gel indicating a polyclonal

regeneration of the recipient livers (Fig. 4A). All vector-genome junctions located closer than 500 kb to the TSS of annotated selleck chemicals llc see more genes were included for analysis of clonality. Using LM-PCR we aimed to identify expanded cells and clonal imbalance rather than the full repertoire of insertions. The limited input of DNA for LM-PCR (0.5%-1% of total liver cells and 10% of initial DNA for nested PCRs) and the coverage using three enzymes for genome fragmentation (76.5% as determined by capture-recapture analysis (Supporting Fig. 7) reduced the overall number of detectable insertions. Based on our calculations (Supporting Fig. 6) we expected to recover around 150-200 insertion sites per repopulated

mouse liver. Averages of 109 ± 25 and 142 ± 84 unique insertion sites per liver were allocated in the in vivo and ex vivo groups, respectively (Supporting Table 4). The mean vector copy numbers of 1.70 ± 0.24 per liver were similar in all generations of serially transplanted mice (Fig. 4B). To determine potential clonal selection after serial transplantations, we calculated the number of clones contributing to 50% of all 454-reads per liver. The contribution of top 50% (T50) clones in latest-generation livers was not significantly reduced when compared to first-generation livers, either in the in vivo group (10.6 ± 1.2% and 11.6 ± 2.2%, P = 0.686) or in the ex vivo group (13.9 ± 1.4% and 12.9 ± 2.7%, P = 0.806), respectively (Fig. 4C).