I would propose lipofection. E.g. see Aoki K et al., Cancer Res. 1995 Sep 1;55(17):3810-6 or Guo XZ et al., Oncol Rep. 2005 Jul;14(1):59-63.

Hi Stefanie,

I would do transfections with the empty vector containing G418 resistance and titrate the amount of DNA, transfection reagent, and G418 concentration. PCR of the G418 cassette rather than your gene of interest (GOI) would be better means of assaying transfection efficiency.

Another option is to do what we are currently trying in our lab. We are using a vector that has a fluorescent protein cloned downstream of our GOI. An Internal Ribosome Entry Site (IRES) is cloned between the two proteins and allows independent expression of the two proteins. Thus positive transfectants will fluoresce and this will allow application of microscopic and flow cytomtery techniques. Here is a link:

https://www.takarabio.com/products/gene-function/fluorescent-proteins/fluorescent-protein-plasmids/red-fluorescent-proteins/dsred-express-and-dsred-express2-fluorescent-proteins?catalog=632540

Terry

How long did you monitor your cells after addition of G418? You stated that after 10 days they all died but in my opinion it could just be that you didn't give them enough time to form clearly visible resistant clones. Usually after 7-10 days of G418 treatment the cells seem to have all died, but then few days later it is possible to see clones. The time necessary to see them of course depends on cell lines. I would wait 3-4 weeks before stating that they all died.

Cecilia Bucci Thank you for your answer. Most of my colleagues pick colonies after 2 weeks. I waited around 2-3 weeks. Do you see clones or small colonies when you pick the cells?

If your colleagues pick colonies after 2 weeks with the very same cell lines then there must be something wrong either with the plasmid you use or with your transfection procedure. I suggest you to check transfection efficiency first (you could use a GFP expressing vector so that you can easily count the percentage of transfected cells) before trying again the experiment. If transfection works (you don't need high transfection efficiency for this, even 10% will be OK) then I will use control plasmids. In particular, I suggest a pilot experiment with: a) an empty vector without the resistance gene b) the empty vector you use c) the empty vector used by your colleagues c) the vector with your insert. From the results you should have a clear idea of what it is going on.