stable eukaryotic transfection. - Neomycine resistance and bad viral titer (Oct/20/2005 )

hi, we used for cell immortalization 2 viral vectors pBabe hygro hTert and pBabe neo Tsv40.
We use electroporation to transfert these ones into empackating cell (PT67). We obtained several transfected PT67 clones with antibiotics selection.
We meet 2 problems:

1) In non-tranfected PT67 cells or pBabe neo- tranfected PT67 cells some clones are resistants to our usual 400µg/ml Neomycin quantity. When we increased neomycine (800 µg/ml) these clones don't died. We have the some problem with others cells lines. In our previous assays to evalue neomycin quantity all (100%) the PT67 killed in 8/10 days with 400 µg/ml. We don't understand this antibiotic resistant.

2) When we test viral suspension (from the tranfected PT67) on NIH3T3 cells to determine the TITER we have not positif results (= any NIH 3T3 Clone resistant to 400 or 800 µg/ml of neomycine).


thank you for your help
Pascal

So what is your question?

I think your transfection efficiency is the issue here. You may want to check with a GFP expressing plasmid. As far as neo resistant colonies, over time you may see a very isolated incidence of such a thing, but usually this is after a long period of selection.