In article <2704251228101994.A66895.VENUS.118AE3190000*@MHS> PINTO at CCC.MEDCOLPA.EDU ("Angelo J. Pinto") writes:
>Can anyone give me any helpful suggestions on how to grow a
>high-titered HSV-2 pool that is pathogenic in mice?
The procedure is not, in itself, particularly difficult. Things
to remember are that mouse strains differ dramatically in their
susceptibilty to HSV, and also that HSV-2 strains rarely grow to titres
as high as HSV-1.
There are a number of HSV-2 strains you can use. HSV-2(333) has
worked well for me. Infect subconfluent Vero cells with a low MOI (say
0.01 - 0.1) - we usually started with, say, 1 x 10e8 or more cells (in our
case, 10 - 12 150mm tissue culture dishes). Incubate under standard
conditions for 2 - 3 days, untill CPE is advanced. You want to catch the
cells just before they start to lift off. Discard the medium, scrape the
cells off and spin them down; the vast majority of the virus is
cell-associated. Resuspend into 5- 10 ml medium. Freeze-thaw the cells,
then sonicate them - we did 50% power, 3 x 15 seconds or so, interspersed
with periods on ice. This should give you titres of around 5 x 10e8/ml.
Ian York (york at mbcrr.harvard.edu)
Dana-Farber Cancer Institute, 44 Binney St., Boston MA 02115
Phone (617)-632-4328 Fax (617)-632-2627