Production and Purification of Lentiviral Vectors

Lentiviral vectors are efficient genetic tools for neuroscience research. Among several kinds of lentiviral vectors, human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors have shown promise. Envelope glycoproteins studding the surface of viral particles play a crucial role in viral infection of host cells. Conventional lentiviral vectors are pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G), and efficiently transfer the gene to different neurons. Lentiviral tropism can be manipulated by genetically engineering glycoproteins. Analyses of specific neural pathways are essential to understand brain functions controlled by complex neural circuits. Viral vectors ferrying transgenes via retrograde axonal transport provide a powerful tool for the analysis of neural pathways. We developed efficient lentiviral vectors for retrograde gene transfer in diverse neural pathways in animal models: highly efficient retrograde gene transfer (HiRet) and neuron-specific retrograde gene transfer (NeuRet) vectors. Both vectors are pseudotyped with fusion glycoproteins composed of rabies virus glycoprotein segments and VSV-G segments. HiRet/NeuRet vectors have been extensively used in neural pathway analysis. In this chapter, we describe the experimental procedures we developed for producing and purifying HIV-1-based VSV-G and HiRet/NeuRet vectors; we also present helpful points for conducting experiments smoothly and effectively.