Transfecting a cancer gene - (Jan/13/2011 )
I'm working on transfecting a cancer gene (a fusion of two normal genes) into cells. It's known to be very transforming on it's own and so I was guessing most of the cells I was transfecting would turn cancerous. This was not the case. Using stem cells, they ALL die. This result is fine as a result is a result. But I need to be sure that it is the gene that is killing the cells and not because the vector comes from a dodgy prep containing accidental trizol or something.
So, I want to find a cell line that can support the cancer gene. What would be the best cells to start with? I've heard people mention so many different types of cells I don't know where to start. IS there a classic cell line that can take whatever is thrown at them? Or is it possible that my gene is so transforming it won't go into anything? It must be supported by something as it appears in the human body.
Any suggestions are welcome, thanks.
I see some contradiction in what you are saying... your transforming gene doesn't transform? How many cells did you transfect? Even genes like HPV-E7 only transform about 1 cell per 1,000,000 in transfections. Could it be that you missed the transformation of a few cells? - all you would get would be some small colonies that may not survive very long without other cells around.
How much DNA are you adding? Can you consistently transfect the stem cells with other genes? What are your controls? Wouldn't it be simpler to make a fresh prep of your gene and try transforming that?
Technically pretty much any transformed cell line should be able to take the gene, though dosage may have some effect. You probably don't want to use 3t3 - they are mouse. I would start with HeLa or HEK293, which are very robust and widely used for transfections. the American Type Culture Collection (atcc.org) has a full list of the cells available if you want more info.
Could it be toxicity that comes with your transfection? Do you have any control group that can tell apart that this is not due to your gene?