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Calculating MOI of viral vector


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#1 philman

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Posted 21 January 2011 - 02:45 AM

Right I am making a lentiviral vector containing my gene of interest, E-selectin, with the aim of infecting a cell line and creating a cell line which will constitutively express this gene. My min problem is now on calculating the concentration of viral particles I have in my supernatant and subsequently calculating the MOI of my virus.

The person showing me the technique for making the lentivirus uses a vector with the GFP protein in, and simply calculates the MOI by seeing the % of cells that fluoresce green under microscope. However I do not have GFP in my lentivector as I wish to use a FTSE labelled antibody later on to examine surface expression of my protein.

Does anyone else have any ideas for calculating MOI or concentration of virus in my supernatant, I have heard that it is possible to do so via RT-qPCR as well but I wouldn't know how to do this. I was also thinking of simply titrating various concentrations of my virus sample onto my cells and measuring how nay of them now express my protein via flow cytometry to get a rough estimate of how much of my virus sample would be required to cause most of the cells to express the E-selectin.

So does anyone have any ideas?

#2 protolder

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Posted 23 January 2011 - 10:29 PM

Hola, Iīm not of the lentivirus field but I work in Baculovirus. There is a method by RT-PCR for titering (Clontech), itīs fast and you have the titer in 4 h. but for me is a bit expensive. If you are able to see the difference betwen infected and non infected cells, I use the end-point dilution, where you seed 100ul of a suspension of 10e5 cells/ml in each well of a 96 wells plate , inoculate 10ul of each dilution of virus in each file or column. Incubate and see the plate each day after infections. In the wells where the dilution is low symtoms of infection will appear soon while were the inoculum is low in virus takes more days to see infected cells , and where there isnīt virus the cells get confluence. Counting infected and non-infected wells you can approximate the titer, but for an exact data there are some excell sheets to calculate as the Reed and Muench calculator. I hope have to hope you. Buena suerte




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