Choice of vector linearisation site for electroporation

[Kevin Mulcahy]

Hi,

I have been trying to stably transfect the monocytic leukaemia cell line 
U937 with a cDNA cloned into vector pCR3 (Invitrogen). However, out of 
30 G418-selected clones (800µg/ml) none of them expressed the cloned 
cDNA so I was wondering if anyone had any ideas as to how I can get 
expression of the cDNA in this cell line.

The method that I am using is electroporation so I linearise the 
vector-cDNA construct before performing the electroporation. The enzyme 
that I use is Dra III which cuts in the F1 origin of pCR3. This cut is 
223bp upstream from the start of the CMV promoter which drives the 
transcription of the cloned cDNA. Is it possible that this is too close 
to the CMV promoter so that when the construct is inserted into the 
genome the cDNA is not transcribed?

Or is it more likely that stable expression of this cDNA in these cells 
is deleterious and therefore only cells which do not express this cDNA 
survive? (We have no idea what the translated protein does). To test 
this out I am going to try and stably transfect these cells with another 
cDNA cloned into the same vector to see if the cells can stably express 
a cloned cDNA in my hands (according to the literature this has been 
accomplished by many other groups). This new cDNA is definetely not 
deleterious to the cells and therefore if expression does not occur then 
the linearisation site may be at fault.

If anyone has any other ideas I would be very grateful to hear them.

Many thanks,

Kevin Mulcahy.