You’ve spent days and weeks thinking of an amazing project. You’ve written your protocols, designed your experiments, and prepared your reagents. You’re going to engineer the best thing since CRISPR; you are ready to clone! But...how?

Sometimes it feels like DNA and protein get all the attention.There are numerous ways to detect DNA-protein interactions or to analyze chromatin states (CHIP-seq, FAIRE-seq, Cut & Run) and to detect protein-protein interactions (yeast-two hybrid, Co-IP, BioID),  and that’s just to name a few. But what if you want to study RNA-protein interactions? The characterization of RNA-protein interactions has lagged behind, likely due to limitations of current means to detect RNA-protein interactions. To address this need, the Khavari lab at Stanford created the RNA-Protein interaction detection (RaPID) method. RaPID borrows the E. coli biotin ligase BirA* from BioID and allows a researcher to identify proteins that bind an RNA motif of interest in living cells.

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.