Abstract
Primary cultured neurons represent a benchmark preparation for studying neuronal cell biology. However, due to their poor transfection efficiency, studies often require virus-mediated gene delivery to genetically alter primary neurons. Lentiviral systems, based on HIV, are a commonly used gene delivery system due to their efficient expression, ease of production, and low toxicity to target cells. A powerful technique in interrogating protein function is ablation of specific proteins from cells using RNAi. In particular, expression of shRNA against the target protein allows for stable, long-term reduction of the protein of interest. Here, we describe a lentiviral system we use to knock down target proteins in primary cultured neurons via expression of an shRNA. We outline the cloning steps required to produce an shRNA-expressing lentiviral construct and provide a detailed method that can be used to produce lentiviral particles to transduce cultured neurons. In addition, we describe how to concentrate the produced lentiviral particles, allowing their use for in vivo studies.
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Acknowledgments
We thank the BBSRC (BB/R00787X/1 to KAW and JMH), the Leverhulme Trust (RPG-2019-191 to JMH), and a Wellcome Trust Investigatorship (220799/Z/20/Z) to JMH for funding this work.
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Wilkinson, K.A. et al. (2022). Using Lentiviral shRNA Delivery to Knock Down Proteins in Cultured Neurons and In Vivo. In: Martin, S., Laumonnier, F. (eds) Translational Research Methods in Neurodevelopmental Disorders. Neuromethods, vol 185. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2569-9_1
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DOI: https://doi.org/10.1007/978-1-0716-2569-9_1
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