MCMV culture and in vivo infection questions - (Aug/04/2009 )
I'm planning to do in vivo virus infection on mice. my first step is to use murine cytomegalovirus (MCMV).
I read a lot of paper they have the virus isolated from salivary gland extract of infected mice. This procedure maybe too technical for me. I'm not sure I can do well in the dissection. Do you think cell supernatant (e.g. 3T3 cells infected with MCMV) is good for infection by injection too?
Secondly, I have problem in making cell supernatant with high titer. Can anyone suggest some tips in preparing MCMV virus stock from cell culture?
Thank you very much!
There are plenty of papers out there with in vivo experiments with MCMV using tissue culture virus, salivary gland virus is better but not always essential, I guess it depends on your experiments and what you are hoping to see.
It is not very difficult to dissect out the salivary glands from mice, is there no-one who could show you?? Are you the first person in your lab to work with MCMV??
The key things for making a high titre stock are to make sure that you are infecting your cells with a fairly high MOI to begin with. If this means starting out in, say, a well of a 12 or 6 well tray then expanding your virus stock up- then that is what you will have to do. If your infected cells take too long to reach 100% CPE (more than 5 or so days) then you will get a fairly low titre stock, as virus will have begun to go bad. Ideally your infection should come down in 3 or 4 days. If you have not reached 100% CPE by then, I would freeze/thaw and then inoculate your supernatant onto a fresh batch of cells to try and increase the titre.
You can also pellet your virus from the supernatant by high speed centrifugation and resuspend in whatever volume you like.
Hope some of this helps