Transfection of HAEC cells

[Knut Tomas Dalen]

Have anyone stabile transfected HAEC cells ?

I have transfected HAEC cells with lipofectin-reagent and a pEGFP-N1 vector ( 
green fluorescent protein ) and found out that the transfection methode worked. 
5 -10 % of the cells were fluorescent. 


Then I tried with my plasmid.
Short after transfection all cells seemed normal. Two days after transfection 
100 ug/ml medium geneticin ( G-418 ) were added. After 8 days, only a small 
fraction of the cells were alive. I then concluded that these living cells had 
been transfected.
Since the wells contained very few cells, I decided to let them grow before 
splitting, and just added new selective medium. Two days later I found out that 
also these cells were dead.

My questions are :
- Is it possible that the "active" concentration of geneticin gets higher, when 
  the number of cells falls ?
- Should the concentration of geneticin be lower in the first place ?
- Have anyone stabile transfected these cells before ?

We are also working at another project, where the transfection efficiency must 
be higher ( not stabile transfection ).
Have anyone transfected HAEC ( or other endothelial cells ) with high 
efficiency ?


Thank you very much for your answer

Knut Tomas Dalen
knutd at biokjemi.uio.no