can i use antibiotics with lipofectamine - my cells have antibiotic as selective marker and need neomycin (Jan/08/2009 )
I have a problem that i am only after eealising now despite knowing i had to this this step for the last two months so any advice would be great
My problem is i am using lipofectamine (have to use this as no other transfection reagents available to me) to transfcet my cells today with a luciferase reporter gene
The cells i am transfecting today are cells which were immortalised a few months back with pSV3-neo which has a selection marker for neomycin
As i was unsure of when (if ever) to remove the neomycin fully from my growth medium my cells have just been cultured since then in 50 ug/ml neomycin in growth medium (after selection for one week in 100 ug/ml) and a paper for similar cells said they cultured their immortalised cells for up to 6 months in neomycin!
The problem i have, which i only realised today, is that when doing the second transfection i need to remove all antibiotics from the medium, i am fine to remove penn/strep but i think now i should also remove the neomycin as it could stop the lipofectamine working
As i have always cultured them in neomycin i am worried that the removal could have some unwan ted effects on the cells, has anybody had this problem before and does anybody suggestions on what i sjhould do?
Will removing the neomycin now have an effect on the cells?
Should i have removed it from the media alot earlier than now?
Thanks for any suggestions
EDIT: (sorry when i say i have neomycin in my media i mean G418 as selection agent)
It's fine to grow your cells for a bit without antibiotics. The G418 in the media helps to maintain the 'purity' of your cell line in the long run. But removing it for transfection won't adversely affect the cell metabolism in any significant way. They certainly don't need G418 to survive, it just so happens that they can survive in media that contains it, and non-transformed cells can't.
So for the transfection with lipofectamine, I'd use antibiotic-free media, but you can continue to grow the cells in G418 after you've completed your transfection experiments.
thanks ginger for your help, i pressumed for such a short time it wouldnt have had an effect but i was not sure and did not want to chance it without some advice,
on the removal of g148 in general, my cells have been passaged up to about 10 times now do you think it is ok to remove the g148 for good now, is there any benefit to keeping them exposed?,
i know you said it keeps the purity which is what i have read in other papers but removing at this stage wont allow other (lets call them dormant cells) which havent grown at all so far to all of a sudden start to grow now that the g148 is removed?
G418 doesn't just arrest cell growth, it kills them, so after 10 passages the number of non-transformed cells in you culture should be pretty much zero.
The reason people use G418 in the media for routine passage of transformed cells is to maintain selective pressure for gene expression. One problem when creating transformed cell lines is that the host cells have a tendency to eventually shut down expression of experimentally added transgenes. They do this by methylating the transformed DNA that has been integrated into the genome. So you can detect the transgenes by PCR, but the cells won't be expressing the proteins you want. Obviously, this is bad for your experiments.
However, so long as you keep G418 in the media, the only cells that can survive are those that still are actively expressing the transgenes that were introduced to the cell.
To give an example, the transformed cell lines I use for viral packaging have been stably transfected with genes for viral envelope proteins and neomycin resistance in one huge DNA cassette. If I grew these cells in media without G418, I would not have any easy way of knowing if the genes encoding my viral envelope proteins were being expressed or not. If the transformed DNA was completely shut down by methylation, the cells would still grow normally, and I'd only discover the problem when I started to have trouble packaging viruses.
However, by maintaining the cells in G418, I have a much better guarantee that my cells will be expressing my proteins of interest: the only cells that will survive in culture are those that are expressing the genes for G418 resistance, and if the gene encoding G418 resistance is being actively transcribed, there's a good chance that my gene encoding viral envelope are being transcribed as well. It's not a 100% guarantee, but it's probably a 80% or 90% guarantee, which is plenty good enough.
I hope I've made this clear.
PS-Hopefully, now you can also now understand why it's not a big deal to remove G418 from the media for a few days. Your transgene expression won't be significantly reduced during this time, but if you leave G418 out of the media in the long term, then you'll start having problems with your transgene expression being permanently and irreversibly shut down.
Yes ginger you have made it very clear for me,
As my original transfection was performed to insert a plasmid which would immortalise cells (by binding to p53 and RB) rather than for the expression of desired proteins which are needed for a certain function, although i know my plasmid works in the same way, i suppose i could easily tell if the removal of G418 has had an effect on the cells
Beacuse if it does the removal from the cells should result in the cells no longer being immortalised and thus they will die
In all cases it is probably best for me to maintain the use of G418 at the low levels or withdraw it gradually, and by permorming cell counts and viabilty assays determine the effects on the cell numbers and growth rates compared to control which would finally give me the answer to the effect of the removal of G418