Hello everyone,
I have a question concerning viruses, again, because I am having some troubles.
I had checked the titer of my lenti with a p24 assay, finding out that I have something like 10^5, when I was more expecting a 10^7 or more. I am using the protocol of a previous colleague that now disappeared, and she has always claimed she had enough viruses.
A colleague told me that I can use the viral supernatant (so the newly produced virus), to re-infect new cells and increase the titer.
But now I really only have a problem understanding how this works: if when I transfect my cells, I have to use three plasmids (one for the gene of interest, one for the packaging and one for the envelope), if I incubate my cells with the viruses, do I have the whole virus again when I collect the supernatant?
Is there anyone that can suggest me articles, papers, assays where I can study the very basics of how this works? Or can you explain it to me?
Because I can only think about cells integrating the DNA of my gene of interest, but I cannot think about them re-producing the whole virus.
Hi Giulia,
On Addgene site you can find the Lentiviral Guide (https://www.addgene.org/viral-vectors/lentivirus/lenti-guide/). It's very useful and easy to follow. This guide is good from the basics to the advanced level.
Here you can read an another very good paper about the lentiviral systemArticle Nucleic Acid Delivery: Lentiviral and Retroviral Vectors
You have said that you use 3 plasmids, therefore this is a 2nd generation system. The 3rd genetartion system is more safer. All of the required plasmids are available from Addgene site (https://www.addgene.org/) also.
After the co-transfection of the virus propagating host cell line (e.g. HEK 293 or HEK 293-AD) with the 3 plasmids then lentiviruses had assembled in host cells and the ready viruses has escaped into the cell culture media.
You can use the supernatant of this cell line without concentration also. But i suggest to split the SN into smaller single use aliquot followed by freeze at -80°C. Avoid frequenty freez-thaw cycles. Becuse every cycle can decrease the virus titer by 25 %.
You should concentrate your lentivirus. No need expensive equipment for virus islation neither if you use the PEG precipitation protocol despite of ultracentrifugation.
To answer your quetsion after transduction your assambled viruses (which not incorporated into the target cell cytoplasm followed nucleus integration) has been in the SN of the target cell line. But you should consider that these environment impure. It consist off the target cell line's by-products. It can be acidic. There can be many dead cells in it. Contaminated with the target cell line dead cells. So I think you should use fresh virus aliquotes (propagated earlier) despite of this SN to re-transduct any other cell line.
I have been used Reverse Transcriptase Assay to check virus titer. I havent't used the p24 Antigen Assay which is very common. Here you can read a paper about titration method comparison.Article Comparison of lentiviral vector titration methods
I have some experience in lentivirus (3rd generation) propagation and transduction. If you need any help just feel free to write! I have got detailed protocols for the entire process (from transduction to clone screening)
Bests,
Tamas
I don''t think when you reinfecting the cells will increase virus titre. Never do this, it will only reduce the titre in the supernatant. Only way you can increase the titre is by optimizing your plasmid ratio of all the three plasmids. generally 4:2:1 (Sh:packaging:envelope) works but you can always do some permutation and combinations.
Tamas has given you some great resources here, however the heart of your problem is that your titre is low (or your p24 assay isn't working I guess).
Your colleague may have been producing a different virus. It's not uncommon for different lentiviral plasmids to produce vastly different titres depending on the transgene, size, generation etc. What virus are you trying to produce?
Apologies for the self-promotion, but I wrote an article about it which might be helpful: https://bitesizebio.com/28063/best-improve-lentivirus-titer/
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