Thanks for all the replies to my original message. In response to the
suggestions to see if pHebo can efficiently stably-transfect the
parental COS-7 cell line (i.e. non-pREP9-transfected COS-7), I agree
totally since it may be that pHebo may not be able to do this. I have
now set up a hygromycin titration on COS-7 to obtain a 'kill curve" and
thus the concentration of hygromycin to use for selection.
In terms of the hygromycin concentration that I am using for selection
of the elusive "double-transfectants", I use 200µg/ml which is
reasonably high. This was determined on the pREP9-transfected COS-7
cells using a 2-week incubation in a 24-well plate with concentrations
of hygromycin B ranging from 0, 25, 50, 100, 200, 300, 400, 500, 600,
700, 800, 900 and 1000µg/ml and a cell-split of 1 in 15 (i.e approx 6.7%
confluency) into the plate wells. Since 200µg/ml was the lowest dose
which killed every cell in the well, do you think it may be better to
increase the selection dose of hygromycin to say 300 or 400µg/ml in
order to make it more stringent?
Another point is that I can't even see any evidence of cell division
following splitting into selection medium. Cells that survive the first
24 hours of selection in hygromycin B (plus 500µg/ml G418) just adhere
to the bottom of the wells and just sit there without dividing until
they eventually die and float off (most of the cells die in the first 24
hours of selection which was not what occurred when I performed the
original selection of the pREP9-transfected COS-7 cells in G418). Thus,
do you think that the cells may have lost either the pREP9 or pHebo
plasmids during the 48 hours post-transfection growth period (i.e.
before the hygromycin B was applied) since the cells may not be able to
sustain very high copy numbers of OriP-based vectors? If this is likely,
how soon after transfection would you impose the hygromycin selection
(the method of transfection is Lipofectamine)?
Many thanks again,
Kev.
