In my previous experiment, I used 4T1 cells to develop stable cell line. The vector I used is pBudCE4.1, Zeocin (500ug/mL) as the selection maker. The size of entire plasmid is 8.2kb. When circular plasmid DNA was employed to do transfection, the transient transfection efficiency in 293T cells is more than 90% 48 hours post transfection, and 25% in 4T1 cells by a polymer transfection reagent.However, I did not observed any cell colony in 96-well plates 14 days post limiting dilution under Zeocin selection neither with circular nor linear DNA. Does anyone successfully generate stable cell line with 4T1 mouse cancer cell under Zeocin selection? Please give me some suggestion.
Are you sure that your problem is the transfection/stable integration or could it be that the problem is the selection? 500µg/ml seems quite high, did you do a kill curve before for your cell line?
Hi Ariane Eberhardt~Did you mean to calculate an IC50 concentration of Zeocin?
Dear Li Sheng Chang,
I was talking about checking the concentration of Zeocin you need for your specific cell line. Because every cell line tolerates of course another concentration of antibiotics and you have to test the right concentration before.
Normally you seed wt cells and add your selection antibiotic in different concentrations, change medium every two days for 7-10 days. The right concentration is the lowest one that kills all the cells.
Because if you use a high concentration then it may be that also your positive clones are dying if the concentration if toxic for every cell.
Dear Ariane Eberhardt, Before the selection, I have treated wt cells with Zeocin at 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000ug per ml for 10 days. And I used an ATP based detection kit to determine the lowest concentration that kills all the cells. Maybe this detection kit is too sensitive for this purpose.
I think this kit should work... did you use the empty vector as transfection/selection control? And could it be that your gene of interest ist killing your cells when overexpressed?
It is a nice advise, I indeed did not include an empty vector control to verify whether my gene of interest kill the host cells. Thanks for your suggestion!
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