Success Transfection but no Lentiviral Production?

22 November 2023 2 9K Report

I've been having issues with viral production for the last couple of months. I was able to successfully make lentiviruses using a 2nd generation system 3 months prior and have a few aliquots frozen, but have unable to make more viruses. We recently tested the frozen nonconentrated virus by transducing a leukemia cell line and our transduction efficiency is about 80-90%.

My current protocol is:

Day -1) Seeding 293T on a 10 cm2 plate.

Day 0) Check to see if 293T confluence is 70-90%.

Prepare transfection complex:

Dilute packaging (psPax2), envelope (pRD114a) and transfer plasmid at 1:1:1 molar ratio in optimem.

Dilute PEI at 3X DNA MW in optimem.

Incubate for 10 mins.

Aliquot the PEI into the diluted DNA mixture. Incubate for 20 mins.

Add PEI:DNA mixture to 293T.

Day 1) Change media with fresh DMEM + FBS.

Day 2+3) Harvest supernatent. My transfection efficiency is about~80% evident by my GFP/mcherry reporter gene.

When I attempted to transduce the same leukemic cell lines, I was unable to detect my fluorescent reporter. It seems like even though my transfection was successful, my 293T are just not packaging the virus. Its not our transduction method because we are able to transduce with our old virus stock just fine. I tried different envelopes, different transfer plasmids, different aliquots of the packaging plasmids, freshly thawed 293T, different incubators, and different FBS manufacturers (both HI and non-HI). I could try different base medias incase the DMEM lot is bad, but i'm not sure if thats the case. I don't think it's out PEI because our transfection has been working well.

Aarya Chitransh

t seems like you have performed a thorough investigation and troubleshooting of your lentiviral production protocol. Given the efforts you've made, let's explore some additional suggestions to help identify and address the issue:

Check Plasmid Integrity:Confirm the integrity of your plasmids, especially the packaging (psPax2), envelope (pRD114a), and transfer plasmids. Perform restriction digests or sequencing to ensure that the plasmids are in good condition and have not undergone mutations.

Optimize Transfection Reagents:While your transfection efficiency seems good, consider trying different transfection reagents. Try a commercially available transfection reagent specific for lentivirus production. Lipofectamine 3000 or calcium phosphate transfection may be worth exploring.

Evaluate Cell Viability:Confirm the viability of your 293T cells on the day of transfection. Healthy cells are essential for efficient viral production. Try different passages of 293T cells and ensure that they are not contaminated.

Optimize Confluence at Transfection:Experiment with different confluence levels at the time of transfection. Sometimes, altering the confluence of cells at the time of transfection can impact lentiviral production.

Check for Mycoplasma Contamination:Perform mycoplasma testing on your 293T cells. Mycoplasma contamination can affect cell health and viral production.

Evaluate Transfection Duration:Experiment with varying the duration of transfection (e.g., 48 hours instead of 72 hours). In some cases, a shorter transfection duration may be more effective.

Consider Changing Antibiotics in Media:If you use antibiotics in your cell culture media, consider changing or omitting them. Some antibiotics can affect lentiviral production.

Virus Concentration and Titering:Check the viral titer and concentration methods you are using. Confirm that the concentration step is not causing loss of infectivity. Try different methods for concentration or directly use non-concentrated virus for transduction.

Evaluate Cell Passage Number:Try using cells at lower passage numbers. High passage numbers can sometimes result in reduced transfection efficiency and viral production.

Use Different Cell Line for Packaging:Consider using a different cell line for lentiviral packaging, such as HEK293FT cells. This cell line is specifically designed for high transfection efficiency and viral production.

Assess Media Quality:Test a different batch of DMEM or switch to another base media. Ensure that the media components are not affecting viral production.

If, after trying these suggestions, you still encounter issues, it might be beneficial to consult with colleagues or experts in your research community who have experience with lentiviral production. They may provide additional insights or share their own successful protocols. Additionally, collaborating with a lentivirus core facility could be an option if the problem persists.

Yuanyi Wei

You could also try reaching iut to a company that specialize in lentivirus production. That could save a lot of time. We always use LipExoGen for any kind of lentivirus we need. They have the most variety and also work realky great :)

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