Demystifying and simplifying the lentiviral production and transduction workflow

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Lentiviral workflows are common in research labs that work with a variety of gene delivery related applications, including gene and protein expression, T-cell engineering, cell line generation, and therapeutic model generation. However, due to the complexity and length of the process, researchers may not always be up to date on the newest techniques and technologies for the most efficient workflow to follow.  

In addition, labs oftentimes rely on multiple providers for the products needed in each step, creating several variables that need to be optimized which can complicate an already time-consuming and complex process. By understanding and simplifying the lentiviral workflow, you can streamline your lab processes and enhance the overall performance of your lentiviral transduction experiments.  

A standard lentiviral workflow 

The most common lentiviral workflow includes five key steps that are essential for successful gene transfer and stable expression.

1. Vector selection and gene cloning: Select the right lentiviral vector backbone to carry your gene of interest. Vectors should not only work with your target cell type but also help improve transgene expression, virus titer, and overall function. Next, clone your gene of interest into the vector. To speed up the process to just a 15-minute reaction, tools like In-Fusion seamless cloning can help clone PCR fragment(s) into linearized vectors.
2. Virus packaging and production: To produce the lentivirus, co-transfect the lentiviral vector—containing your gene of interest—with packaging plasmids encoding essential viral genes into a packaging cell line. Systems like fourth-generation lentiviral packaging, with optimized ratios of transfection reagent and packaging plasmids, can help produce very high titers while improving safety. 
3. Harvesting and titration: After harvesting the lentivirus, it is critical to titrate your virus to determine the amount of supernatant needed for your desired multiplicity of infection (MOI). Quantification can be easily completed in just ten minutes with lateral flow assays. Kits are also available for ELISA and qRT-PCR-based lentivirus quantification.
4. Concentration and/or purification: Depending on the titer, there may be a need to concentrate the lentiviral supernatant to increase the viral particle count in the transduction mix. This helps ensure efficient delivery to your target cells. Commercially available concentrators can help achieve 100-fold concentration of the lentiviral supernatant. In case of sensitive target cells, a purification step can help remove any cytotoxic contaminants.
5. Transduce the target cells: Introduce the viral particles to the target cells via transduction. While this step is the most critical and can be difficult to master, innovative and easy to use products can help transform your transduction experience.

Once all steps of the lentiviral workflow are complete, you can determine whether it was successful by analyzing your gene’s integration into the host genome.  Techniques such as PCR,  Western blotting and integration site analysis kits can be used to confirm the success of your workflow.

Simplifying lentiviral transductions    

For those new to lentiviral production and transduction workflows, its numerous steps—the transduction step in particular—can be overwhelming. Fortunately, recent innovations are making it simpler and more efficient. A transduction sponge, for instance, allows you to follow an easier transduction process:  

Lenti-X™ Transduction Sponge workflow

In addition, the transduction sponge enables your lab to transduce 10⁵–10⁷ cells across a variety of cell types, including difficult-to-transduce primary cells. It even eliminates the need for harsh chemicals and minimizes cell handling, offering a non-toxic approach that boosts transduction efficiency without the need for labor-intensive methods like spinoculation. Altogether, innovations such as this allow both new and seasoned researchers to focus on their downstream experiments rather than an otherwise complex lentiviral workflow.

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A simpler lentiviral workflow can save your lab significant time and resources. Go a step further and download a free lentiviral workflow infographic poster, so you have a readily available resource whenever—and wherever—your researchers need it.