Vector gene copy number and histochemical staining of skeletal muscle of C57Bl/6 Rag-1-deficient mice 14 weeks following injection of AAV vectors encoding F.IX-WT or variant K5A/V10K. (A) Southern blot analysis of genomic DNA extracted from murine skeletal muscle injected with AAV-F.IX-WT or F.IX-K5A/V10K at the doses indicated. Copy number standards were prepared by adding 3.5, 35, and 350 copies of plasmid per murine diploid genomes. (B) Immunofluorescent staining for human factor IX in tibialis anterior muscle. Animals were injected in the hind limbs with 1.2 × 10¹² vg/kg AAV-F.IX-WT or K5A/V10K divided equally among 6 injection sites. Representative sections from the injected muscle are shown. Excitation of fluorescence tags revealed both intracellular and extracellular signals in muscle injected with F.IX-WT, but markedly reduced extracellular signals in muscle injected with F.IX-K5A/V10K, while intracellular signals are clearly detectable. Original magnifications, × 200.