peter.cherepanov at uz.kuleuven.ac.be
Tue Mar 5 03:32:34 EST 2002
I do not understand what can go wrong here... it looks fine to me,
may be the right thing to do in this particular case is to check for CD8
expression by your phoenix cells.
They are designed so that normally one can judge MLV gag-pol expression by
looking at CD8 levels. I hope you do not have to order expensive antibodies
just for that....
Also, try infecting other murine cells. With retroviral vectors there is a
big difference between different cell lines for the transduction efficiency
(promoter plays a big role too, of course).
... adding polybreen ... (a modest increase in transfection).
If you can get your hands on Phoenix-gp cells and pMDG to envelope your
virus with VSV-G, you can get as high titers as you want by concentrating
your virus (simple pelleting by ultracentrifugation).
The last comment - actually _do_ select your transduced cells and watch
colonies. I my experience, you need much less resistance gene (G418 or
hygro) expressed to have a great colony then to see LacZ or GFP expression
(never used Gluc, sorry). I bet you will see lots of colonies. If you
generate a library to look for a complementation of a mutation or for a
particular phenotype, you might need just a very modest expression of the
gene. The expression here is very much determined by the site in the
chromosome were your provirus ended up, not only by promoter you used.
(Myself, I only used P.-ampho cells)
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