alphaTC1-9 expression after 1 month - (Jun/19/2008 )

hi guys, i transfected my vector into some alphaTC1-9 cells. i know my transfection worked based on immunofluorescence microsopy (roughly 5% of cells were transfected). however, following one month of G418 selection, it appears the selection process did not work since the percentage of cells expressing the vector were not any different from the percentage seen on day 0 of transfection. any ideas what might have happened and how i could improve this process?

here is what i did specifically: 600 ug/ml G418 for two weeks, then 400 ug/ml G418 for a week and then 800 ug/ml G418 for an additional week.

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1. Did you select and expand the clones or grew a pool? You need to select well expressing clones.

2. Do you know from literature or killing curve, whether 600ug is high enough for these cells? For most of my cells it is 1mg for two weeks, once I select the clones, it is 400ug/ml

3. If you have transfected a fluorescence gene, you may add FACS to your selection strategy. Just collect the top1% expressing cells and expand. The expression may still go down with this strategy, best is G418 clone selection-> western for your protein-> expansion of best expressing cells.

hth/

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600 ug/ml G418 is enough because after 1-2 days following application, rounded up cells were visible. for the next two cells, viable cells were very, very low.
however, at the two week mark, there was a dramatic increase in the number of cells almost over night, which i thought was strange. when i stained for IF microscopy at the end of the month, it turns out the selection had not worked.

i am selecting for a pool of cells btw.

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