By now you have probably heard about Addgene’s viral service but did you know that Addgene also performs independent research? Since launching the viral service in 2016 Addgene’s research team has been actively investigating ways to improve viral vector production. Whether redesigning viral packaging plasmids, investigating different viral production strategies or developing novel methods for data analysis, our team’s goal is to provide scientists with new tools and protocols that they can easily incorporate into their own labs. This week we are pleased to announce the submission of the results of our first study which can be accessed here at bioRxiv.

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.

The central nervous system (CNS) orchestrates complex processes enabling organisms to control their movements and behavior. These functions and others are controlled by collections of neurons that are intricately wired into circuits through synaptic connections (Shepherd, 2004). Understanding the structure and function of these neural circuits is essential for neuroscience research. The use of genetic tools for visualizing and perturbing circuits together with the development of methodologies to deliver genes to the CNS have recently made it much easier to map these neuronal networks.

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.

While lentiviral vectors are popular gene delivery tools, producing lentivirus, can pose certain challenges. Whether choosing a system that is the best fit for the experiment, trying to produce virus of a usable titer, or fine-tuning selection and expression in your target cell line, researchers often find themselves faced with a roadblock. In this post, we will provide an overview of some of the common challenges associated with producing and using lentivirus and offer some tips and tricks for overcoming these hurdles.