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?


Thanks you for any suggestion!

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

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.

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.