293T cells are already G418 resistant. Perhaps you are working with these, and not the original/parental HEK-293 cell line. You can run a qPCR to look for expression of the NeoR gene to determine this if your records are ambiguous as to which version of the 293 cultures you have.

In addition, G418 is not the most stable drug. Make sure you are using newly reconstituted antibiotic, and that your stock has not expired. Old G418 will not work well for selection.

With which HEK293 cells do you work exactly? If it is the HEK293T subline, then this will not work, since the stable transfection of the T-antigen also added a resistence against G418 (and related antibiotics).

You can either try to get the original (untransfected) 293 cells or switch to something else.

Hello Daniel!!!

You are saying that 293-T are different from HEK-293 and that the first one are naturally resistant to the G418, and it's possible that I am selecting a resistant cells??? At the beginning I did the kill-curve with untrasfected cells and I saw that died even if not all. I have already use in the past this cell line (in a different lab) and I didn't have any problem; now I received this cell from another group so I am not completly sure that is a parental cell line.

I was also thinking to do a western blot and if I will see a great difference between trasnfected and untrasfected cells just continue.......!!!! What do you think, is not the best method to remove any doubt???

I was also thinking to use neomycin, even if it should be the same.....!!!!!

I am using a lot of G418 and I am buying two bottles for month from two month already.

Nope, you are not selectiing resistant cells, you are using resistant cells. So its no wonder that your controll cells are not dying.

And its pretty possible that the cells you used in the past, were the original 293 cells, the cells you have now are most likely the 293T subline. Most people are not aware of this difference.

If you intend to go on working with these cell, i would recomend you to do a cell line autentification (commercially available at DSMZ or ATCC for example), to be shure what cells you are using. and some journals already ask for this, when you submit a manuscript.

best, Guido

Unfortunatly you are right!!!!!!!! I asked for the HEK-293 and just few minutes ago I discovered that the cell I am using are 293-T, so I just lost my money and time, convinced that I was using the other one....!!!! I can't do anything to use this cells, for example change the substance even if my plasmid pCMV6-AC-GFP has the neo-gene. Maybe I have to start again a new selection!!!!!

If some of your cells are still GFP positive, you can FACS sort for GFP. If not, or you don't have access to FACS, you'll need to start from scratch.

Also, in the future, you might try blasticidin or hygromycin resistance cassettes, if possible. These act faster to kill off sensitive cells and cell lines rarely harbor resistance to these markers due to prior selection.

Hello Daniel, I transfected my cells with the mutation form of the gene and the wt; while for the mut form I started from a big colony (even if there were few untrasfected cells) and in my flask now I have a lot of green cells with some untrasfected, for the WT the situation is different; I have the 70% of untrasfected cells and the rest part of green cells.

So I still have green cells in my flasks, obviously the problem is the presence of the untrasfected cells.

Thank you

Be careful with GFP. This gene has a quite long half-life for the mRNA (if I remember correctly about 18 hours), the protein itself is even more stable. So even if your cells are green, they don't necessarily still express either your transgene or even GFP. I wouldn't waste more time and money and start over. Then you know for sure that your cells are clean and you can properly select for your transgene. I know that this is a hard step, but sometimes this is necessary (I have gone through this before). Good luck.

I would try to FACS sort for single cells that are GFP positive. It sounds to me that you have enough positive cells to get stables for both the WT and mutant. Even 30% positive is more than enough to try FACS with an easy to propagate cell type such as 293T.

I have a doubt: if this cells 293T are resistant to the G418, even if I added the selection substances, they dind't undergo a selection pressure necessary to keep the gene of interest so it's possible that the cells lost the gene....!!!! It is just a thought!!!!

What do you think?

I thought that your transgenic cells are GFP positive. Was the GFP a separate control? If your transgenic cells are indeed GFP positive, then I don't see how they would lose the gene of interest. If I misunderstood and they are GFP negative, then please disregard the FACS suggestion and start over with parental HEK-293s.

As a negative control I have had onlyt the untrasfected cells; I dindn't trasnfect with the only empty vector as a positive control. I have extracted the proteine from one flask (70% green more or less) so I could do a western blot for my proteine and compare with the untrasfected cells and if the expression will be fine I may continue doing the FACS as you suggested. Could be a right decision?

Yes, if the cells were transfected two months ago and are still positive for your transgene by Western blot, I think you could purify stable cell lines by FACS based on a GFP marker.

It is impossible to select 293 T cell with Neomycine. 293T are neomycin resistant because of the presence of a neomycin resistance cassete together with the SV40 Large T . So you should have still viable cells in culture even after adding huge amount of neomycine. if not, this means that you 293 are not 293T cells.

Thank Patrik, I think that the cell line that I have udsed was 293T and not HEK-293. There was a mistake, because I asked for the second one, Just to say, I was trying to select with G418. Anyway, I managed to obtain that cell line.

Thank you for your interesting!!!

Gm

Gianmichele Massimo 

would you please tel how much is your G418 concentration for HEK-293 CELLS

Hi, 

are you referring to the concentration for selection of for maintaining? If you are using immediately after transfection, 24h later, start with an high concentration like 600-700ug/mL, for the first 24-28h, and then you can move down to 400-500ug/mL for usually three weeks more or less. I suggest to check your cells routinely, and wait for the appearance of single colonies. Then pick up single colonies and move in a well of a 96/well plate. Once confluent, move cells from each well into a well of 48 well-plate and so on for 24, 12, 6, and 25cm2 flask. So, before to expand one specific line and use for your experiments check the expression profile  of your protein of interest by any quantitative method....let say western blotting. Choose the colony with the highest grade of expression and expand as normal. 

Once you have a stable cell line, to maintain the expression of your gene of interest keep the cells exposed to G418, 250-300ug/mL, is usually an acceptable concentration. 

Hopefully this help you

Gianmichele

Hi

could you please send me the article for HEK 293 neomycin selection concentration?

Thanks.