Molecular analysis of the mice receiving injection of rAAV/DLZ6.

A. Diagram of primers designed for detection of rAAV DNA. B. Distribution of rAAV in mice following portal vein injection. A unique 450 bp fragment of rAAV/DLZ6 was amplified to detect distribution of rAAV after hepatic injection. DNA isolated from brain, spinal cord, muscle, bone marrow, heart, lung, testis, lymph node, kidney, intestine, and spleen were analyzed following sacrifice of a mouse receiving high-dose rAAV/DLZ6. Liver/LD and Liver/HD refer to DNA PCR analysis of animals receiving low- and high-dose rAAV/DLZ6. Standard curve 5, 1, 0.2, 0.1, 0.01, and 0 genome copy equivalents of plasmid pDLZ6 diluted with genomic mouse DNA. C. Diagram of the primers designed for RT/PCR. D. RT-PCR analysis of total RNA isolated from control and experimental animals. Primers were designed to amplify a 534-bp BDD-hFVIII specific fragment. –RT control employed RNA isolated from the mouse liver receiving high-dose rAAV/DLZ6. The negative control used RNA isolated from control animal. RNA samples of muscle, brain, lymph nodes, testis, kidney, and spleen were from the mouse receiving high-dose rAAV/DLZ6. LD: liver RNA isolated from mouse receiving low-dose AAV/DLZ6. HD: liver RNA isolated from mouse receiving high-dose rAAV/DLZ6. E. Sph I digestion of Diagram of the restriction digestion using Sph I. F. Southern blot analysis of high molecular weight genomic DNA and Hirt DNA isolated from experimental animals. Standard curve: genomic DNA from control mouse liver with 5, 1, 0.2, and 0.02 genome copy equivalents of plasmid pDLZ6 per cell, respectively. HMW genomic DNA and low molecular wt liver DNA (HIRT) isolated from animals receiving high-dose rAAV/DLZ6.