Stable clone formation....please help - (Apr/26/2007 )

Hi everyone,

I am having a real problem creating stable clones and I do not know what is wrong. I am sorry for the long message but I have wasted so much time trying to get this to work....

Here is what I did:

I transfected a plasmid with my protein of interest tagged to GFP into T47D and MCF7 cells using Lipofectamine 2000 in a 1:5 ratio. The next day, I saw what looked like alot of GFP cells in both cell lines. I began selection with G418 two days later. I used a concentration of 350 ug/ul. I had done a kill curve and 350 seemed to kill these cells (or make these cells sick) over two weeks. I saw that, after selection, alot of cells grew in the T47D cell line but not many in the MCF7 cell line.

I checked GFP and it seemed that in both cell lines the majority (if not all) the GFP was lost. Additionally, the cells started to grow in a sick manner (even the empty vector) as I propogated them so it was not that there was just a diminishing of signal with selection.

I assumed that I was just not using a high enough concentration of G418 therefore I repeated the experiment but this time I selected at three different concentrations of G418 (400, 450, 500) I found from my kill curve that 500 killed cells very rapidly so I figured this would work.

Again, I saw the same thing. Many T47D's (without GFP) but very few MCF7 (also lacking GFP). My T47D control plates all died, but I saw some MCF7's growing at the lowest concentration. I searched the plates and found a few colonies that seemed to be glowing green in the T47D plate and MCF7 plates. I carefully picked these clones and tried to propogate them up for a month. They grew very slow but seemed healthy. Unfortunately, I found out today that all my clones were dying or sick and the GFP signal is gone.

I have asked around and I have been adviced to start all over again with a new vector. Has anyone else had this problem?

Thank you very much for any guidance. I am very frustrated.

Cellsgrow

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I've only heard that selected cells have less bright GFP than transiently transfected cells.
I have also tried to clone cells transfected with EGFP tagged protein with hygromycin over a month and
also lost my GFP cells, but that was not my main project so I did not have time to fully troubleshoot!!

I hope someone with the experience can give some advice on this!!
Sorry not much help.

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Yes, stable cell lines normally will have less bright EGFP signal compared to transiently expressing cells.
Your protocol sounds OK. What vector are you using? You sure that your protein won't have bad effects on the cells (like toxic or so) since you said that later the cells look sick? Normally after finish selection you could maintain clones in lower concentration of G418. How do you check the GFP signal? Just by looking under microscope or do you do IF? Looking at live cells under microscope may not let you see the signal but in fixed cells you will have better chance. You can try using anti-GFP even to see how. Have you tried induced them (with Nabutyrate for example?) to see if you still have the signal? In some cases the signal is very weak, but will be enhanced with induction. The clones that you found seems to be glowing green may be dying cells. I have found that dead cells could glow much brighter than real living stable cells.

Anyway, I normally don't use stable clones but stable pooled cells. So after transfection, I do FACS for GFP expressing cells and then select with G418. After 2 weeks, I do FACS again. If I do stable clones, I normally don't choose just green dots but pick lots of surviving colonies into individual wells, continue selection or maintaining until they grow enough to do scanning by IF to choose real stable cell lines.

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