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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.

It was by serendipity that I got into the field of gene therapy, more specifically AAV-based retinal gene therapy. The year was 2001 and I started a job as a technician in a lab using adeno-associated viral vectors (AAVs) to treat an inherited retinal degenerative disease called Retinitis Pigmentosa. I quickly became fascinated by this emerging technology and its potential for the treatment of some genetic diseases.

As part of our partnership with the Penn Vector Core, we will be expanding our inventory of tools for calcium sensing. In this post, we’ll review the main categories of sensors we’ll have available.