Abstract
Top of pageAbstract It has been observed that DNA-dependent protein kinase (DNA-PK) inhibits adeno-associated virus (AAV) DNA integration in vitro and in vivo (Song et al PNAS 2004). In order to test the effect of DNA-PK on AAV replication, we have infect MO59J (DNA-PK negative) and MO59K (DNA-PK positive) cells with a recombinant virus (UF5) in the presence or absence of a helper virus (carrying AAV rep and cap genes). Hirt DNA were isolated 2 days after infection, and subjected to southern blot analysis. Data showed that the abundance of replicated monomer of the vector DNA was lower in MO59K cells than in MO59J suggesting DNA-PK may inhibit AAV replication. To avoid the effect from the difference other than DNA-PK between the two cell lines, we have employed the small interfering RNA (siRNA) technology to decrease DNA-PK expression in MO59K cells. Synthetic siRNAs for human DNA-PKcs were transfected into MO59K cells. Total RNA were extracted at 2 or 4 days after transfection. RT-PCR analysis showed that DNA-PKcs mRNA was nearly undetectable after 25, and 35 cycles. These results indicate that siRNAs can efficiently target DNA-PKcs, thus provide a powerful tool for creating cell or animal models to study the cellular effects on AAV replication and integration.
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