To determine whether rAAV had disseminated, DNA was extracted from the optic nerve, brain, lung, liver, kidney, testis, heart, spleen, skeletal muscle (m. rectus femoris), and blood. The tissues (except blood) were homogenized (Biotron, Göttingen, Germany), and genomic DNA was extracted from each tissue according to the manufacturer’s instructions (Stratagene). These organs were obtained from rats that received transplants of AAV-LacZ-IPE cells, AAV-BDNF-IPE cells, or IPE cells alone or age-matched control rats with direct injection of AAV-LacZ, AAV-BDNF, or vehicle. The concentration of DNA was quantified by spectrophotometry (Ultrospec 4000 UV/Visible Spectrophotometer; GE Healthcare, Tokyo, Japan), and the DNA concentration of each organ was equalized to 100 μg/mL.
Blood was drawn from the tail and collected in tubes containing sodium heparin. Genomic DNA was isolated using an automatic DNA purification system (Genextractor TA-100, together with the Genextractin Kit; TaKaRa). PCR was performed as described.
24 Two sets of primers were used; the first set was 5′-TGGAGTTCCGCGTTACATAAC-3′ and 5′-CCGCATCACCATGGTAATAG-3′, which amplified 323 base pairs (bp) of the AAV vector promoter region (CMV); and the second set was 5′-AGAGAGGGAGTGGCCAACTC-3′ and 5′-CATGACCCGTATTACGGTCC-3′, which amplified 359 bp from the AAV inverted terminal repeat (ITR) region.
The PCR amplification conditions for the first primer set were: 95°C for 10 minutes; 35 cycles at 95°C for 1 minute, 57°C for 30 seconds, and 72°C for 2 minutes and an extension at 72°C for 7 minutes (GeneAmp PCR System 9600; Applied Biosystems, Kanagawa, Japan). The amplification conditions for the second set of primers were: 95°C for 10 minutes; 35 cycles at 95°C for 1 minute, 56°C for 30 seconds, and 72°C for 2 minutes; and an extension at 72°C for 7 minutes.
Then, 0.5 μL of genomic DNA was added to 10 μL of PCR reaction solution including 1.0 μL of 10× buffer (ExTaq; TaKaRa), 0.80 μL of dNTP mixture (2.5 mM each), 0.5 μL of each primer, and 0.05 μL of polymerase (5 U/μL; ExTaq; TaKaRa), and 6.7 μL of distilled water. The PCR products were separated by 1.5% agarose gel electrophoresis, stained with ethidium bromide, and viewed with a UV transilluminator.
For the positive control, we extracted DNA from the retina after injection of AAV-LacZ into the subretinal space on the indicated days. For the negative control, we examined the DNA from untreated retinas. The methods were same as described earlier.