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Since Addgene began producing AAV vector preparations, we have used quantitative PCR to evaluate the physical titer of our preps. We often repeat this assay to confirm that our results are accurate. But what if we could repeat this assay 10,000 times to ensure that our titers were as accurate as possible? This is where droplet digital PCR (ddPCR) comes in.

Understanding the biology behind brain development, learning, and behavior poses many challenges in neuroscience with many brain regions, neuronal networks, and specific neuronal populations to consider. To tease apart how different neurons impact a certain output, neuroscientists have begun to use more precise genetic tools: activators, inhibitors, sensors, recombinases, and more. With the help of adeno-associated viral vectors (AAV), neuroscientists can combine classical genetic tools with the manipulation of specific neuronal subtypes and determine how these changes affect the phenotype. Viral vectors and genetic approaches are continuously refined and developed so there are many combinations to choose from for your experiments!

Dear scientists using AAV,

We hear your questions! How much virus should I inject? How long do I need to wait? What serotype and promoter should I use to target my favorite neurons?

We don’t know all of the answers, but we’re betting that your colleagues do. That’s why we’re launching the AAV Data Hub. The Data Hub is an open platform for scientists using AAV to share practical information about their experiences. It will contain all of the juicy and important details that you won’t find in the final publication and we have already collected over 100 entries spanning six different species and dozens of different expression sites.

Since rolling out our viral service in 2016, Addgene has shipped thousands of AAV and lentiviral preps to scientists around the world. In the years since, our scientists have optimized many steps in the viral production and quality control process to improve both the yield and quality of the viral preps. For example, last year we published an article describing a sucrose supplementation strategy to improve yield. In addition, as described in this blog post, the team developed a viral genome sequencing strategy to confirm the identity and purity of all AAV viral lots.

Standard AAV vectors are generated with a producer cell line like the widely used 293T. During vector production, assembled AAV vector particles accumulate inside the producer cells and purification protocols often describe a process that begins with cell lysis to release AAV particles with subsequent purification steps. It’s also possible for AAV vectors to be spontaneously released from the producer cells into the supernatant during the production phase. However, this has not been well characterized as it is not a common event.