Double-transfection (stable) of COS-7 cells

[Kevin Mulcahy]

I have recently stably-transfected COS-7 cells with cDNA cloned in the 
eukaryotic expression vector pREP9 (Invitrogen) which confers resistance 
to G418/geneticin and is maintained in cells as an episome (since it 
contains the origin of replication for EBV "OriP" and also expresses the 
EBNA-1 gene necessary for vector replication via OriP). However, I have 
been trying to transfect the "pREP9-transfected" COS-7 cells with 
another cDNA cloned in another plasmid which confers resistance to 
hygromycin. Such a double-transfectant should obviously be resistant to 
both G418 and hygromycin and possess both pREP9 and pHebo as episomal 
plasmids due to the expression of EBNA-1. However, I seem to be having 
problems with this double transfection.

This second vector "pHebo" is similar to pREP9 in that it possesses 
OriP, but it confers resistance to hygromycin (however, it does possess 
the EBNA-1 gene). I was trying to think of possible reasons as to why it 
is proving very difficult to generate the double transfectant. One 
reason I thought of might be that the hygromycin resistance gene of my 
plasmid may not be expressed in these cells. However, the hygromycin 
gene in pHebo is driven by the same promoter as the G418 resistance gene 
in pREP9 so you would expect the hygromycin-resistance gene to be 
expressed also.

Another possibility may be that the cells were not able to support the 
episomal maintainance of two different plasmids with the same origin of 
replication? Does naybody know if this is likely in mammalian cells?

Apart from these two points I can't think of any other reasons why I 
don't seem to be able to create a double transfectant. In terms of the 
cells, they were extremely easy to transfect with the initial pREP9 
(20-30% efficiency of tranfection using a B-gal reporter plasmid) and 
the actual pREP9-transfectants are extremely easy to transiently 
transfect too (again, 20-30% efficiency of tranfection using a B-gal 
reporter plasmid). The hygromycin titration (to obtain the appropriate 
dose of hygromycin for selection of double-transfectants) was performed 
on the "pREP9-transfected" COS-7 cells in the presence of G418 so I 
can't see why the selection conditions would be incorrect.

Therefore, I am at a loss! Does anyone have any other ideas as to why 
the double transfection may not be working? I hope so.

Many thanks,

Kevin Mulcahy.