Double-transfection (stable) of COS-7 cells

brett brett at BORCIM.WUSTL.EDU
Fri Oct 25 10:52:31 EST 1996


>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.

Dunno. Does your pHebo vector work in non-"pRep9-transfected cells"? This
may address whether there is an incompatibility of the origins. You might
also consider utilizing the SV40 large T antigen present in COS-7 cells to
replicate your plasmid from an SV40 origin. Good luck.



Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

"I own my own pet virus. I get to pet and name her." - Cobain




More information about the Methods mailing list