Hi everyone!
I want to transfect my cloned plasmid vector into thyroid cancer cell lines (8505C, B-CPAP, K-1, MB-1 and BHT-101). Can anyone suggest me the best way to do the transfection and advise me with protocol?
I need to select the successfully transfected cells by G418. Could you please also suggest me the concentration of G418 and incubation time for the selection process.
Thank you very much.
Lipofectamine is quite good at transfecting cells. However, if you need to make it really working use electroporation: http://www.nature.com/nmeth/journal/v3/n1/full/nmeth0106-67.html . Each cell line can have a very different sensitivity to G418. It is recommended to titrate each cell line before using for the real selection.
Usually around 500 ug/ml For start screen between 250 and 750 ug/ml.
We tend to use lentivirus expression systems as they transduced with high efficency into many cell types both cancer cell lines and primary.
I use lipofectamine, works great. You should also decide if you want transient (temporary) or stable (permanent) tranfection. For transient, recombinant plasmid is used, while for stable, you need to linearize your plasmid before transfecting. Select a restriction enzyme that will cut outside your gene in only 1 place. Clean the linear plasmid to get rid of proteins and then use for transfection. You may have to try different G418 concentrations, I use 300 ug/ml to 400 ug/ml depending on the cell line. Incubate for at leat 1 week to 10 days. Treat untransfected cells at the same time to make sure your cells have endogenous responsiveness to G418, that ay, if your recombinants survive, you very likely have positive transfectants.
Dear Toks Yerokun, thank you very much for your advice. Do I need to always linearize cloned plasmid before transfection? The protocol I am following is not suggesting to linearize cloned plasmid before transfection. If I transfect my cloned plasmid without linearise it, is it possible to get stable cell line by this way? Advice me please. Thank you very much once again.
Linearizing increases the chance of your gene being integrated into the genome of the cell. The presence of your gene as a recombinant in theDNA ofthe cell is what makes it stable transfectant. Transfecting the circular vector results in transient transfection because, eventually the cell will eliminate the vector after several rounds of growth. although you do not have to linearize the vector in all cases, however, after some time, everytime you want to do an experiment with previously transfected cells, you will need to use newly transfected cells, unless you have tested to confirm that your gene is still being expressed by the previously transfected cells.
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