Hey all,
I am attempting to transiently transfect three plasmids into CHO cells. My plasmids are a CRE-luciferase (minimal promoter), pRL-Tk-Renilla, and my gene of interest driven by CMV. On my first attempt I used a 5:1:5 ratio of CRE-luciferase, renilla, and my GOI (50 ng: 10 ng: 50 ng). When I analyzed the results of my luciferase assay I saw that both my luciferase and renilla showed poor signals. I am wondering if the amount of my GOI is somehow preventing the expression of my two reporter genes. Is it simply a matter of toning down the dose or is there some other nuance with my transfection? I am using lipofectamine 2000 and am successfully able to transfect the plasmids singly and am also able to transfect the CRE-luciferase and Renilla simultaneously.
Dear Sushanth
Which well format are you working in? I persume 96 well plates based on your quantity of DNA.
I would try to increase the amount of DNA input, while monitoring your transfection induced toxicity. I think that you could try to double the amount of DNA (at least in HEK293T cells this should work). We use a 2:1 ratio of Lipo 2000 when we increase our DNA input, as most of the toxicity will be cause by the lipofectamine and decent expression is maintained.
Good luck
Philippe
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