I am trying to create c2c12 stable cell line. I performed death curve and found 1mg of g418 was optimum for 95 % of cell death and at 1,5 and 2,0 mg no survivors at all. Then I performed nucleofection of my plasmids and after 24 hours, I trypsinized and seeded them in media containing 1mg of G418. I got only false positive survivors.
I repeated the experiment again by increasing the concentration of g418 to 1,5 and 2.0 mg along with 1.0 mg . In all conditions am getting only false positive survivors (Not jus a single colony but lots of colonies) . I seeded cells very diluted way (250 000 cells in 150 mm culture dish). So they are not close to each other as well.
How the cells got resistance to survive in such high concentration of g418. Even if there is no transfection (which is not, I checked a batch of the transfected cells under microscope after 24 hours) how they are able to overcome the resistance.
Hi Mohammed!
Before to go further I have a naive question - are you sure you DNA prep you are using for transfection is not contaminated with empty vector? Also how to you assess expression of your protein? By WB?
I am sure the plasmid I use free from contamination, I have created stable cells using the same plasmid in other cell lines (like HeLa), and I did Immunofluorescence on c2c12 cells before selection to check if they are expressing on the right place. Did western blotting on other cell lines after transfection and got band at right molecular weight. Though the transfection efficiency is 30-40% percentage, I dont know why there is no integration in the DNA of the host and also how the untrasnfected survives in g418.
Hi Mohammed,
Since you checked your plasmid, there is one more possibility left-I guess!
In some cases, there is hypermethylation of the plasmid's promotor get rid of it as one of cellular protective strategies. One option is to change the promotor of the plasmid (for example, if your plasmid is CMV, try put your insert in another plasmid with EF1a or U6). Because transcription machinery differs between cell types.
Please try to check if any one before did successful transfection for any gene and used a plasmid with a specific promoter and follow the rule.
All the best,
Zakaria
Ok than few more points to consider:
1. Concentration of G418: you should use the LOWEST concentration as possible, i.e. the concentration which basically will kill all control cells within a week or two (not faster). You do not want to have all your cells dead after 48h, it is not a good point.
2. When transfecting a circular plasmid, it will be randomly cutted before to be eventually integrated into the genome. I believe your Neo gene and your insert are under different promotors. It happens that only the Neo gene gets integrated, but not your insert, which could explain why you are getting those false positive clones. Also, the longer is your insert, the higher the risk to be cutted - you can try to linearized your plasmid outside of the insert / Neo / promotor region before to do the transfection.
3. If nothing helps, keep in mind that G418 is easy and commonly used but not necessarily the best selection - maybe consider using another selection...
Thanks for your suggestion. I performed death curve for a week and found 1mg G418 was optimal. Since the cells are surviving in 1mg, i increased upto 2mg to check if it will help or not. I will try linearising the plasmid, even if that fails as you said I will go for different selection.
Hi Hakim,
I faced a similar issue with C2C12 cells. I tried a couple of things of which seemed to help:
a) I start selection always 24 hours post transfection. Even if you trypsin treat and re-seed the cells, start selection only after the cells attach.
b) I have a suggestion/comment: Make sure that the G418 stock concentration is dependent on the active component of the G418 and not total salt. Eg. in Gibco G418, out of every 1mg total G418, only 700ug is the active component. Accordingly, if you think you're adding 1mg/ml, you might be actually adding 700ug and then your kill curve numbers change.
Refer Table1 on this link: https://www.lifetechnologies.com/at/en/home/life-science/cell-culture/transfection/selection/g418.html
and finally,
c) Use a vector which has Puromycin selection. It works wonders and was the best solution to the problem. 1ug/ml Puro works well with C2C12s and you can start seeing cell death after 24 hours. G418 selection is very very slow, it takes about a week or more to see some death and it is still very heterogeneous in results.
Good Luck!
Utkarsh
I have had this problem. Are you adding the G418 to an entire bottle of media and letting it sit for a few days in 4 degree and then warming it? If so, the G418 might be loosing it's potency. Try adding it to each flask or dish individually. Also, make sure to wash off all of the G418 before mixing any of the cells with trypan blue as the two together cause a precipitate that will skew your cell counts/viability. Hope this helps.
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