Lentiviral workflow

When choosing a plasmid backbone, you have many features to consider for expression of your gene of interest. Constitutive or inducible expression? Untagged or fluorescently labeled? Explore available options to find the best vector for your experimental aim.

In-Fusion cloning is ideal for inserting your fragment into a lentiviral vector—just one quick reaction allows you to recover your final construct.

Learn how In-Fusion Cloning was used to quickly clone PCR fragments into a lentiviral vector that had only one suitable restriction site for subcloning.

The most critical factor for successful lentiviral transduction is viral titer. Start with our fourth generation packaging system and obtain between 10⁷and 10⁸ infectious units per ml, 25 times what other popular systems generate.

It is important to have an accurate estimation of infectious forming units (IFU) in your packaging supernatant. This measurement will determine how much supernatant is used to achieve a desired MOI—and ensure successful gene delivery. There are 4 titration kit options available from Takara Bio:

1. Need a fast answer? Lenti-X GoStix take only 10 minutes to assess lentivirus titer and determine whether your supernatants are ready for harvesting.
2. Use an accurate ELISA-based protocol to measure p24 capsid protein content in your supernatant.
3. Use qRT-PCR to determine the viral genome content in your viral supernant from a calibrated RNA standard curve.
4. Quantify the number of lentiviruses that have integrated into the nuclear DNA of your target cells (provirus) using a green intercalating dye-based qPCR reaction.

For more information see the lentiviral titration kit selection guide.

If your IFU/ml titer is low, Lenti-X Concentrator can be used to concentrate your virus up to 100-fold and improve gene delivery. This concentration method is scalable, easier, and faster than ultracentrifugation.

Transduction problems can be frustrating and waste valuable resources—inhibitors in your media or difficult cell types can hinder lentiviral gene delivery. Learn how to navigate transduction troubles.

Want to avoid Polybrene and speed up transduction? Bring virus particles from your lentiviral packaging supernatant and cultured cells together with charged magnetic beads. Some cell types, such as hematopoietic cells, show very low transduction efficiency even with high-titer lentivirus. RetroNectin reagent promotes co-localization of virus and cells to dramatically enhance transduction efficiency.

The life cycle of lentivirus results in integration of a copy of the lentiviral genome into the host genome. The chosen integration site has important consequences for both the expression of your transgene and the phenotype of the host cell. Identify the specific integration site.