How to improve the transduction efficiency for lentiviral vectors?

23 July 2015 6 1K Report

Hello,

Could anyone share his experience for how the transduction efficiency of lentivectors may be improved. We have used the spinoculation, protamine sulfate addition. Also just an incubation without anything (just medium). Unfortunately the percentage of expression of GFP is not that nice as expected (it's usually 1-2% comparing to untouched control). Are there any special tricks?

Thank you!

Tom Masi

Hi Mark,

I assume you know the titer of your GFP virus? Have you tried using different MOIs to transduce your cells? Different cells require different MOIs to acheive a certain transduction efficiency. You may need to use MOIs of 10, 20, 40 to get the efficiency where you want it to be (I am assuming you want close to 100%?). I just use media plus 8ug/ml polybrene and alter the MOI and I can get pretty close to 100% depending on the cells.

Mark O. Pekarsky

Thank you, Tom. Yes, sure we know the titer and we used different MOIs already like 5, 10,20,30 and 40.

And I am talking not about 100% perhaps, 30% would be enough! And I am transducing T regs as cells.

Tom Masi

Hi Mark,

I do not have any experience transducing T regs so I can't help on that front. How did you determine your virus titer?

Simon N Waddington

If you're using VSV-G pseudotyped vector, you might want to consider a different pseudotype. This reference might be of interest.

http://www.ncbi.nlm.nih.gov/pubmed/24578496

BW, S

Danielle May

If you are still having issues, you could try concentrating the virus via ultracentifugation. You just spin your viral media for 1-2 hours at 50000g, then resuspend in a lesser amount of media (let it sit for a few hours at 4 degrees C, then vortex) and put it back on your cells (with polybrene and/or retronectin). I know it has been a while since you asked the question, but I wanted to add this here in case others search ResearchGate with the same question.

Yu Wa

Dear Colleague,

Improving the transduction efficiency of lentiviral vectors is pivotal for successful gene delivery, particularly in gene therapy, functional genomics, and cell biology studies. Here are several strategies to enhance lentiviral vector transduction efficiency:

Optimize the Vector Design:Incorporate strong promoters (e.g., CMV or EF1α) to increase the expression of the transgene. Use a self-inactivating (SIN) lentiviral design to improve safety and transgene expression. Consider including woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) to enhance mRNA stability and transgene expression.

Concentrate the Lentivirus:Concentrating the lentivirus through ultracentrifugation or precipitation methods can significantly increase the viral titer, thereby enhancing transduction efficiency.

Polybrene/Protamine Sulfate Use:Adding cationic polymers like polybrene or protamine sulfate to the transduction medium can neutralize charge repulsion between the viral particles and the cell surface, facilitating viral entry.

Optimize MOI (Multiplicity of Infection):Determining the optimal MOI for your specific cell type is crucial. Too low MOI can result in insufficient transduction, while too high MOI may lead to cytotoxicity. Pilot experiments to find the optimal MOI are recommended.

Enhance Viral Entry:Pre-treatment of target cells with reagents that modify the cell membrane, such as chloroquine, can enhance viral entry. However, these conditions need optimization to avoid cytotoxicity.

Improve Target Cell Condition:Ensuring cells are in optimal condition (e.g., healthy, actively dividing, and not over-confluent) at the time of transduction can significantly impact efficiency.

Use of Spinoculation:Centrifuging the cells together with lentiviral particles (spinoculation) at low speeds (800-1,000 g) for 1-2 hours at room temperature or 32°C can improve transduction efficiency by increasing virus-cell contact.

Temperature Optimization:Performing the transduction at 32°C instead of the standard 37°C can enhance viral fusion with the cell membrane, thus improving transduction efficiency in some cell types.

Sequential Transduction:For hard-to-transduce cells, performing sequential rounds of transduction (e.g., on two consecutive days) can increase the percentage of transduced cells.

Remove Serum During Transduction:Temporarily reducing or removing serum from the culture medium during transduction can increase viral access to the cell surface. Ensure to return to normal culture conditions post-transduction to maintain cell viability.

Target Cell Receptor Enhancement:For cell types with low expression of lentiviral receptors, overexpressing these receptors or using pseudotyped lentiviruses that target abundant cell surface molecules can enhance transduction.

Implementing these strategies requires careful consideration of the specific requirements of your experiment and cell type. It's also critical to comply with biosafety regulations when working with lentiviral vectors.

Best regards.

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