Hi Omid,

No problem to do co-transductions with two different lentiviral vectors. It is a routine practice in our lab (human and murine cells) and we have no problem with this type of protocol. You can also do the transuction in two times if you want an offset expression of the two transgenes, it depends of the experimental purposes. Let me know if you have other questions!

Best,

Nicolas

You could go even higher - in our hands up to 5 different lentis could co-transduce the same cell. More than 5 we just did not try, should also work. Everything depends on your titer and MOI. Furthermore, if you need 2 viruses only in a mix you could also produce 2 viruses simultaneously - just transfect HEK cells with both lenti vectors and helper plasmids. If efficiency of expression of viral genomic RNA from both vectors is similar, you will get approximately the same titer for both viruses.

Thanks Nicolas and Konstantin for the feedback. I am experiencing some problems with the selection of dual infected cells with two antibiotics (puromycin and hygromycin B). Is it possible that placing the cells on selection medium with those two antibiotics is just too much for them? I started the selection 3 days after infection and titrated both antibiotics separately in advance...

Although we never tried to select with 2 antibiotics simultaneously, but my guess would be that it should work. Obviously, many single infected cells will die, but still there should be colonies of double-infected cells. I would try separate infection and selection with single antibiotics to confirm that both lentis work.

Hi everyone,

But if you are using the same vector and you infect in 2 different timepoints how do you select the co-infected cells from the single-infected ones, since they must already be resistant to the antibiotic? Do you increase the concentration to select only the ones infected with both?

Thanks for the help.

Hello,

Doesn't work... You have to use two different resistant genes

I am experiencing the same challenges with dual selection with 2 lentis. When I added puromycin first, it was easy to get double resistant clones with subsequent hygro selection. However hygro seems to wipe out the same cells in the same expt. What is the basis for the weak resistance phenotypes yet same titres of lentis were transduced? Why are puro resistant clones easier to get hygro resistant and apparently not the other way round? Thanks

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Hello , can you someone help me providing a reference for the dual transfection of the same cell with different lentivirus vectors. thanks

Dual infection with lentiviruses, also known as superinfection or co-infection, is indeed possible and can be a valuable tool in research. Here are some key points about dual lentiviral infection:

Dual infection can be used to introduce multiple genes into a cell, study gene-gene interactions, or achieve multi-component genetic circuits. It is a powerful technique when conducted with careful planning and controls.

Perhaps this protocol list can give us more information to help solve the problem.