Novel Approaches for Production of Recombinant Adeno-Associated Virus

Publisher Summary This chapter describes how to produce a recombinant adeno-associated virus (rAAV) stock of high purity. Wild-type AAV has not previously been associated with disease in healthy adult humans and is classified as a risk group 1 agent under the NIH's recombinant DNA guidelines (rev. 04/02). Recombinant AAV vectors retain only the inverted terminal repeat (ITRs) sequences from the wild-type AAV genome, with 96% of the DNA genome removed. Add the denatured DNA from the 96-well microtiter plate to wells of the dot blot manifold apparatus in the absence of a vacuum. After all the DNA has been transferred into the manifold, apply a vacuum for 3–5 min. Monitor the dot blot with a Geiger counter. Continue the washes if needed using the high-stringency wash solution. Do not let the membrane dry out or the probe will permanently adhere to the membrane. DNA preparation should be pure. Purify the viral fragment by agarose gel separation and running onto Whatman DEAE-8 1 paper or a preparation of equivalent high quality.