Virus Titration befor infection with Gecko Library

I'm going to use gecko library in order to infect primary macrophages and is the very first time I'm approaching this technique, so I have some questions.

I'm following the nat prot paper by Julia Joung (2017) but I have some questions regarding the titration part.

I'm using the 2 vectors system and I will infect macrophages expressing the cas9.

I red in the paper that infecting a very low MOI is a very crucial step, to allow just one guide (or so) to enter one cell.

My question is regarding how to calculate the MOI: in my system I don't have any reporter, such as GFP or other molecule to track, so I just want to ask if it is correct to do the titration as follow:

- i will transfect Hek293T cells with Pax, VSVG and the library. i will use petri dish, so I scale up to use 16 petri (instead of 4 t225) and 122.4ug from the library.

- after 8hours I will remove the supernatant and replace with fresh medium.

- after 2 days from the starting point, i will collect the supernatant and freeze at -80

at this point i will thaw it to infect my primary macrophages and calculate the titer as follow:

- day 0: seed macrophages at 1x10^5 per well in 24 well plate

- day 1: add to 1ml of medium the virus in 100ul. Make 1:10 dilution: 1:1, 1:10, 1: 100, 1:1000. keep 1 non-infected well

- day 2 add puromycin and leave until the cells in non-infected well result dead

- calulate the titer as: titer: cn/ vv/ mD where cn: number of cells infected at day of infection; vv: volume of virus used for 1:1 infection in ml (100ul) and mD: dilution at last well that showed 100% of infected cells (counted).

Is this method correct?

Final question is: I will use gecko library that contains around 130.000 sgRNAs. I'm planning to infect around 2*10^8 macrophages. Is this a good number?

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Fuchsia Kelpiealmost 3 years ago

Thanks for your interest in our work. To answer your questions:

For lentiviral production, I usually use lipofectamine 3000 and change the media after 4-5h, or you can use PEI without media change. Is there a reason why you are changing media after 8h?

For the titering, you will need to include an additional non-infected well that you do not treat with puromycin. Once the non-infected well treated with puromycin is dead, then the MOI for each virus dilution is the number of cells with virus divided by the non-infected well that was not treated with puromycin. Keep in mind that you will probably want to refresh puromycin every 3 days, and I typically pick the puromycin concentration that kills 95-99% of the cells after 3 days. If this is your first time transducing macrophages, then the wide range of dilutions you are using makes sense, but afterwards you will have to do a finer dilution series (i.e. 2-fold dilutions instead of 10-fold) to more accurately pinpoint an MOI of 0.2-0.3.

The number of macrophages you will infect really depends on how your titer looks. For example, if the maximum MOI you can get is only 0.1, then you will need to transduce a lot more macrophages or concentrate your lentivirus.

Ruby Ghoulalmost 3 years ago

thanks for your kind reply.

I change the medium after 8 hours because I used cacl2 till now to do the infection. But for the screening I will use lipofectamine (2000) so thanks for the tip.

For the titering, i will include for sure the non-infected well with no puromycin but then to calculate the MOI I will do the number of cells with virus divided by the non-infected well that was not treated with puromycin multiply for the dilution factor? (eg: if I'm counting the 1:10 dilution well then I have to do (n cells with virus/ n of cells with no virus) *10, is this correct?

I normally have 99% of cells dead after 4 days in the control, so this should work, and I will use a finer dilution for sure,

Fuchsia Kelpiealmost 3 years ago

If you have not already purchased lipofectamine 2000, I would recommend transfecting with lipofectamine 3000 instead, as we've found that using lipo3000 results in at least 2x higher titer, when we use half the amount for transfection. The protocol for lipofectamine 3000 is posted on this forum elsewhere.

The MOI is calculated as number of cells with virus divided by the non-infected well that was not treated with puromycin, without multiplying by your virus dilution. For instance, you might get a result like:

1:1 virus condition MOI = 0.8

1:2 virus condition MOI = 0.8

1:4 virus condition MOI = 0.4

1:8 virus condition MOI = 0.2

In this case, to shoot for an MOI of ~0.3 you would use the 1:6 virus condition.

The number of macrophages you will need, if you can get an MOI of 0.3 will be (number of sgRNAs in library)*(500 cells/sgRNA)/0.3. You want to aim for the number of cells that still gives you the appropriate coverage, given that 70% of your cells will die after selection.

I do not keep the cells in puromycin after the first 5 days of selection, and I typically screen for 2 weeks after selection. If you are screening at much longer time points, then you could consider maintaining the cells on puromycin.

Ruby Ghoulalmost 3 years ago

thanks so much for the help!

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