All Posts

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!

One of the biggest barriers to gene therapy treatment of central nervous system (CNS) diseases is the blood brain barrier (BBB). It’s the BBB’s job to block the entry of pathogens to the CNS, but this also stops viral vectors, such as adeno-associated virus (AAV), from reaching their targets.

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.

My lab's vector of choice is AAV, with nearly every experiment requiring AAV. Before joining my lab, I had never worked with AAV, so naturally I had to package some virus for my first experiment. It was a bit intimidating, but I had my lab’s protocols and some great co-workers to help me out. Even with these tools, I found myself writing AAV production tricks into the margins of my protocol. While these tips weren’t critical to the experiment, they definitely made my life easier!  In this post, I’ll share some AAV production, purification, and titration tips, while also summarizing the basic steps and analyses needed for packaging AAV.

Background on neuronal tracing

A key aspect to understanding the brain’s function is knowing its architecture, in particular the connections between different brain regions. For example, communication between the hippocampus and the prefrontal cortex brain regions is involved in the formation of episodic memory, a special type of memory which includes autobiographical events (see Jin & Maren, 2015). Directional flow of information between different parts of the brain is mediated via individual neurons. Neurons are composed of a cell body, with dendrites receiving incoming information, and a projecting axon sending information onwards to other neuronal cells. Synapses at the terminals of axons form connections to dendrites of proximal neuronal cells. In the specific example of episodic memory, a subset of hippocampal neurons projects axons directly to the prefrontal cortex, but also indirectly via synapses to neurons in other brain regions. Further, the connections between regions are often reciprocal, forming a neuronal loop which is activated and strengthened during memory formation and memory retrieval.