This protocol outlines simple methods for producing, concentrating, and titrating lentiviral vectors based on HIV-1. Lentiviral vectors are usually produced by cotransfecting 293T cells with plasmids containing the transgene, packaging functions, and envelope (Env) glycoprotein sequences. The VSV-G glycoprotein is often used for pseudotyping, but other glycoproteins can also be used. Pseudotyping refers to incorporating a different viral glycoprotein into the vector membrane, allowing the vector to infect a broader range of cells.

Lentiviral vector titers typically range from 10^6 to 10^7 transducing units per milliliter. For higher titers, physical concentration methods such as ultracentrifugation or precipitation can be used. However, lentiviral vectors with high concentrations of VSV-G can be toxic to cells. This toxicity can be reduced by using alternative glycoproteins or improved concentration methods like anion exchange chromatography or polyethylene glycol (PEG) 6000 precipitation.

There are different ways to measure lentiviral vector titers. Real-time PCR can quantify vector DNA in transduced cells, and ELISA can detect viral proteins like p24. However, these methods might give different results. Functional titration assays, such as flow cytometry to detect reporter gene expression, are often used for more accurate titration.

This protocol includes procedures for producing, concentrating, and titrating lentiviral vectors, offering options for various pseudotypes. These methods help produce higher-quality vectors and accurately measure their titers.

For detailed steps and protocols, please refer to the PDF: