How to calculate MOI based on percent transduction

[Sharp]

Dear all members,

  I'm working on transducting the GeCKO library into mammal cells. However, I was confused when performing the MOI preliminary experiment.

  In Ophir Shalem's articles:Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells, the "Percent Transduction" was mentioned when calculating MOI=0.4. I know that MOI is defined as the ratio of transducing units of viral particles to 

target cells. But in Ophir Shalem's articles,how was the perctnt transduction related to MOI. Which percent transduction was chosen as closet to MOI=0.4? Or you just chose the 30%percent transduction when MOI=0.4?

Hope for the answers,

Sharp

[Neville Sanjana]

Hi Sharp,

In the GeCKO paper, we titrated viruses such that 30-40% of cells survived after puro selection (calculated by comparing surviving cells after puro with transduced cells without puro). Assuming Poisson statistics, this should keep the number of multiply infected cells (ie. cells receiving more than one virus) to a minimum. 

As you point out, percent transduction is the measurable quantity. MOI can be calculated after measuring percent transduction. The terminology can be confusing because for small values of MOI (<<1), percent transduction and MOI have similar values. For a full explanation, please see: http://en.wikipedia.org/wiki/Multiplicity_of_infection

Hope that helps,

- Neville

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[Sharp]

Thank you! It does solve my problem.

Sharp

[Suresh Selvaraj]

Hi Neville,

Can you please elaborate a little on the how you calculate MOI. I dont understand how you compare surviving cells after puro with transduced cells without puro. Is this done certain days after transduction and does it account for cell proliferation?

Thanks in advance,

Suresh

[Neville Sanjana]

Hi Suresh.... Yes, we wait 1 day after transduction before adding puro. Make sure to have both transduced and nontransduced cells and to have both of those with and without puro.

After adding puro, you only need to wait long enough such that nontransduced cells are 100% dead.... normally 2-3 days with puro. Then, count the 2 wells of transduced cells with and without puro. This should get you a % survival, which is what is needed for titering the virus. We usual titer infection for between 30-50% survival after puro selection.

- Neville

[Kenneth Williams]

Hello Neville Sanjana, 

Kenneth Williams here, could you just explain to me how you went from a percent to MOI, apologizes if the answer is painfully obvious. 

Best Regards

Kenneth

[Neville Sanjana]

Hi Kenneth,

We use the standard Poisson model (each infection event is independent).... a full explanation and sample calculation is here: http://en.wikipedia.org/wiki/Multiplicity_of_infection

- Neville

[Deathcrafter 2000]

Do you also calculate transduction efficiency?

Thanks

[Deathcrafter 2000]

For large scale screening is it better to add virus based dilution OR transforming units?

 

I have done some titering in 12 well plates using A549 cells in 500ul media for 24hrs.  The method I used was based on % alive cells after puromycin selection and then calculating how many cells were transduced when I added the virus, and then calculate the transducing unit I have in the concentrated virus stocks eg, if 25 % of cells survived with 0.1ul virus added to cells in 500ul growth media (1:5000), then 2500 out of 10000 cells (cells in wells when virus was added) were transduced, therefore I have 2500 transducing units in 0.1ul virus solution = 25,000 TU/ul.

 

If I take this to large T175 culture where I have 2.5 e+6 cells per flask (I need a lot of cells due to the numbers needed for the library where I will need 26 flasks for each Gecko library) and  transduce in 10ml media.  To get MOI of 0.3  in need   7.5e5 TU ( 2.5 e+6 x 0.3  =7.5e5 TU) , so from viral stock of 25,000 TU/ul I need 30ul virus.  In 10ml media this will be 1 in 333.33 dilution (10,000ul/30ul), which is more concentrated than it was in the 12 well plate titer (roughly 1 :5000).

 

Should I use TU/ul =30ul  or the second dilution method 1:5000 of 10mls that would be 2ul?

 

I'm planning a small scale single flask  T175  optimisation with a control virus to find out.  I've been told by a work colleague that I should use the TU/ul method.

 

Thanks