Hello Chanele,

In order to establish what MOI to use, you should infect a 96-well plate of cells with a titration of your virus. Maybe start at MOI of 100 and dilute down 2-fold, 5-fold, or 10-fold across 5-10 doses. Always include wells with no virus (MOI of 0). Do this in triplicate. Regarding the time of infection, that depends upon the envelope protein expressed on your lentivirus. If it is VSV-G, four hours of infection should be sufficient. You can look in the literature regarding the infection time for your envelope protein. Of course, this will vary based on cell line. You may perform multiple infection time points on the dose response plate if you wish. Also, you should consider whether or not you even need to remove virus. If you are measuring luciferase activity in the supernatant than you should change media and wash after infection and then allow time for new luciferase production and secretion. This is because there may be luciferase in your virus stock from the packaging cell line, which will produce a high background. If, however, you are measuring luciferase activity in cell lysates, then there is no need to change media following infection, as all intracellular luciferase will be from lentivirus infection. If you want to see if there is luciferase in your virus stock, just add some to the luciferase substrate and measure luminescence. Remember that your MOI will likely change with multiple virus stock freeze/thaw cycles, so try to use virus which is at the same storage condition as that which you generate your standard curve with.

Regarding the UC-MSCs in particular, I do not have experience with these. While I do not know the envelope protein on your lentivirus, four hours of transduction should be enough, given that the receptor is expressed on the cell surface. If you want to increase the rate of infection, you may add polybrene. I think that the MOIs which you mentioned should be a good range to test.

Article Human Immunodeficiency Virus Type 1-Derived Lentivirus Vecto...

Good luck!

Sincerely,

David

David Schad Thank you so much for your answer. I really appreciate.

I will try different MOI as you suggested. Thanks.

The envelope protein is VSV-G, therefore I will try to do 4h and maybe 12h and 24h. I don't want to go higher than 24h as I don't want my MSCs to start differentiate.

I was planning to measure the luciferase activity in the supernatant. Therefore I will change the media before I read the plate. What amount of time would you suggest to wait for new luciferase production?

Thanks again!

Best,

Chanèle

Hi Chanele,

Maybe let luciferase produce overnight or for 24 hours.

It will need time for transcription and translation and this should be plenty.

So, change the media to remove virus after 4hr of infection, then wait 15-24 hours and then harvest the supernatants for analysis.

Sincerely,

David

David Schad Thanks a lot again for your answer. I think I'm now on the right track to transduce my MSCs.

Best,

Chanèle

Hi Chanèle Cyr-Depauw ,

I've realized David Schad has given you key suggestions. I've been doing something similar in the past few weeks, and the nature protocol paper below has been my guideline. Take a look at it, and it may guide you step by step!

All the best!

https://www.nature.com/articles/s41596-020-0394-5

Eugene Marfo Obeng Thanks a lot for this suggestion. I will have a look at that paper.

Best,

Chanèle

I have transduced primary mesenchymal stem cells with LentiGlo (ready to use lentivirus expression Gaussia luciferase or red-emitting firefly luciferase, concentration 5x10^7 TU/ml ) form Targeting Systems at an MOI of 30 and seen efficient transduction. ( I used MSCs till passage 4)