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Help: got the stable cell line without my target gene - (Mar/09/2006 )

Hi,

I have tried to make 3 types of stable cell line(piresneo vector), use the piresneoEGFP as control. As this cell type belongs to a stem cell with the potential to differentiation to another kind cell.

During my selection, as the cells died seriously, I grew the poor cells(also my control) without G418 and try to let them recover well. When they grow happy, I increase the con. of G418 to the selection requirement. I selected them at least 10 days, and the cells without transfection died no more then five days.
Each kind stable cell line have been made 7 lines. I harvested the cells for westernblot, unfortunatelly none of them could express my target gene. But I got the piresneoGFP stable cell line. According to the literature, it is impossible to get my stable cell line as overexpression of this protein wil cause the stem cell differentiation. I have to repeat this experiment.
Do you have any suggetion for my depressed results? How can i exclude the possibility that my negative result is not come from my experiment manipulation?sad.gif sad.gif sad.gif


Thanks you for any suggestion! smile.gif smile.gif

-BLUE-SNOW-

your target gene is inframe in the vector, right? did you sequence it?
maybe a southern to check for presence of the construct?
good luck

-aussieuk-

someone in our lab meet the same question,i think you should select more clones to analysis.
if your target gene is in-frame with GFP,it's easy to distinguish the transfected and non-transfected cell.

-bobxiang4-

Thank aussieuk and BobXiang!
My target genes are definitely in correct reading-frame. I can check the expression of those genes in transient transfection samples by western.

Unfortunately, I do not have any GFP in my target gene vectors, and I just transfect the piresneoEGFP to check the transfection efficiency. I can only get the piresneoEGFP stable cell line and never get the stable cell line with my target gene. The problem is when I overexpressed my target gene in cells, the cells will be easily differentiated. Therefore, I can not get enough cells to passage and check with western.

-BLUE-SNOW-