I transfected U251 cells, selected them with blastacidin (20 ug/ml) for 2 weeks and check the expression of the product by qPCR and western. Although the mRNA levels are quite good, I have very little expression of protein. I want to re-select the cells but I don't know exactly what concentration of blastacidin I should use since the cells already have the resistance gene to the ATB. Any tips? Thanks!!
Have you generated a pool of resistant clones or a stable cell line?
Did you do a dose response of U251 cells with blasticidin before you transfected them? How many cells survived selection? You may have to increase the dose to get high expressing cells.
You should select clones generated from a single cell. Dilute cells to the proper concentration and than plate them in a 96 well plate, keeping the blasticidin. in some days you should see colonies. Than you should expand them and check the expression of all the clones: than you could chose the one that expresses more your protein. However, selecting clones takes a lot of time...
Do you still have the mixed population frozen down from the transfection? I like to select my cells at 4-5x the ic100 concentration of the parental cell to get cells with many integrations. Then do your clones in 96 well format. It helps a lot to make filtered-conditioned media from the parental cell then put your selection agent in it when growing your clones out.
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