Double-transfection (stable) of COS-7 cells

[Kevin Mulcahy]

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.