I'm performing transient expression experiments with CHO K1 (suspension adapted). I'm using a pcDNA3.1 vector and PEI and Geneticin for selection. In the protocol I use (Geisse, 2006) nothing is mentioned about using antibiotics for selection after transfection. In literature I only find the use of antibiotics for creating a stable expressing cell line. Is it necessary to use antibiotics for transient expression? If yes, what concentration range should I use to optimize the selection?
Dear Esther,
I have used Chinese hamster cell lines for transient transfection. My vector is pCDNA3 beta with neomycin selectable marker. It can be selected using G418. But for transient transfection I do not select the transfected cells. You do not not to use selection for checking transient expression.
You can get an approximate idea about your transfection efficiency, by using pCDNA3-eGFP as a control. After transfection, I get the estimate of GFP positive cells and transfection efficiency using Flow cytometry. If you get 60% GFP positive cells, you may assume that you have approximately
Dear Esther,
I have used Chinese hamster cell lines for transient transfection. My vector is pCDNA3 beta with neomycin selectable marker. It can be selected using G418. But for transient transfection I do not select the transfected cells. You do not not to use selection for checking transient expression.
You can get an approximate idea about your transfection efficiency, by using pCDNA3-eGFP as a control. After transfection, I get the estimate of GFP positive cells and transfection efficiency using Flow cytometry. If you get 60% GFP positive cells, you may assume that you have approximately
I agree with Tias. I would only add that if you are studying the phenotype of a particular gene you can enrich your positive population by cell sorting.
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