I have been trying to establish a stable cell line using KnockoutTM Single Vector Inducible RNAi System from Clontech. I used Lipofectamine2000 for transfection and then I have selected the cells in 400 micrograms of G418 for 2 weeks. Unfortunately only very less cells survived. When i tried transfection method with different plasmid and it seemed good, however, it was only transient transfection. If anyone tried similar experiment can you give me some advices?
Hi Viola,
For stable lines make sure to linearize your plasmid first. Also make sure to do a kill curve with different concentrations of G418 on untransfected HepG2 cells to find the lowest concentration that kills all untransfected cells. Your transient transfected cells will have many more copies of the resistance gene than your stable cell lines, which might explain why the transients survive selection but the stables do not. Also you might want to sequence the resistance gene in your plasmid to make sure it hasn't picked up any mutations. Good luck!
Have you thought that it might be the transfection? Lipofectamine 3000 can get upto 60% transfection in HepG2. Also, what size is the DNA fragment you are trying to integrate? If it is transfection, having better transfection, will yeild more cells for the selection process, and hopefully more clones in the end. Let me know if you would like more information.
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