I want to select stable HEK with Geneticin in order to express a membrane receptor and run functional studies (overexpression is not important). I read that HEK293Ts are not the best cells for this purpose. Any comments?
Hello Philippe!
HEK293 are abbreviated as 293 and similarly HEK293T cells are termed 293T cells.
293T are 293 cells but were immortalised with the large T antigen and have a Neomycin resistance. Hence, Geneticin (=G418) does not work as a selection marker in 293T cells, as they are already resistant against this. However, Geneticin still owrks in 293 cells. In our lab we have both cell types and currently I am doing a stable transfection into 293 cells with G418 selection.
There also 293TN cells which are usually used as viral packaging cells.
Hopefully this helped a bit. If you have further questions, just ask.
Good luck with experiments and best wishes,
Alexander
If you really want to use 293T itself, U can co-transfect a puromycin resistant plasmid and select for puromycin (1-2µg/mL). The target to puro plasmid ratio should be atleast 7:1. In 8/10 cases I got desired clones with pBABE-puro co-transfection.
If u have your protein fluorescent tagged, its even easier, because u can pick clones in between selection. Obviously u will have some only puro clones, which is minimized by the reduced amount of puro-plasmid.
I have also tried to use extra ordinary high amounts of G418, but is not so efficient .
Good Luck
As explained by Alexander 293T cell line, although easier to handle than 293 cell line, is not suitable for selection with geneticin (G418). Therefore, 293cells are better if your gene of interest i cloned on plasmid carrying resistance to geneticin.
If you would like to use the 293T cell line then you should either re-clone your gene of interest in another plasmid carrying resistance gene to hygromycin or puromycin, which I recommend, or as suggested by Manoj co-transfect a plasmid carrying resistance gene to puromycin or hygromycin.
However, the later method has two disadvantages. First you might end up with cell lines harboring the plasmid with resistance marker but not the plasmid encoding your gene. Second the cells will not have a selection pressure to keep your plasmid and even they will express your gene of interest at the beginning they will loss it after few rounds of selection. So cloning the gene of interest on the same plasmid encoding the resistance marker is better.
I would suggest also that you determine the lethal dose of the antibiotic on the cell line alone by testing different concentrations of the antibiotic on the cell line without the resistance plasmid. This will allow you then to directly apply the antibiotic concentration that kills the untransfected cells but not the one carrying the resistance gene.
Best wishes.
Hi Appalaraju,
Actually this is a well known information about 293T cells because it was generated by transfecting a plasmid expressing the SV40 large-T antigen into HEK293 cells under neomycin selection. The cells were originaly called 293tsA1609neo. As you see the neo at the end of the name indicates the selection marker. However, you can test your cells by exposing the untransfected 293T cells to different concentrations of G418 and check for their survival.
regards
Can't make G418 stables in 293T cells. I've made hygromycin and puromycin stables in them though. Learned they were G418 resistant the hard way, a long time ago.
Hello,
I am working with HEK293T/17 cells now. According to your information, I think I can not use pcDNA3.1 as transfect vector because it is selected by geneticin (G418). I will try a new cell line. Maybe SH-SY5Y
You cannot use pcDNA3.1-neo, but could use pcDNA3.1-zeo. Same vector, except that the selectable marker is zeocin, rather than G418. I have made stable cells lines in 293T cells using the pcDNA3.1-zeo vector and selecting with zeocin.
Hello,
I would like to advise you to always think about which cell line and which vector you want to use before transfection. Try to find out how the cell line is made and which restistant genes are already present. I sometimes get the complaint that G418 is not working on HEK293T cells, but this is normal as by immortalizing HEK293 cells into HEK293T a neomycin resistant gene came in. To solve this problem you can do 2 things:
1) if your vector is already cloned and you have to use G418 for selection, but you have several cell line options, search for another cell line without a neomycin resistance gene to put your vector in.
2) if you really want to transfect HEK293T cells, then you have to clone your gene of interest in another vector containing another resistance gene than neomycin.
I hope this info is useful.
Kind regards
Hi Guys,
Would you please explain also about the suitable concentration of G418 for Killing HEK-293.
Thank you in advance
Kind regards
Hi Elham Khalesi,
Based on the protocols, G418 sulfate (purchased from GIBCO) is normally used at a concentration of 400 μg/ml for selection and 200 μg/ml for maintenance. If you have time, better to make a killing curve for that to get the right concentration. The 293T cells, that we are working, are already transfected and interestingly we could pick up the co-transfected 293T cells in 150 μg/ml. Look at the attached file.
I've made 4 stable cell lines in the last month, in HEK-293's, selecting for stable neomycin-resistant clones. I used G418 at [500 ug/mL]. Worked beutifully!
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