Dear all,
Would you kindly share with me a good protocol for mouse embryonic stem cells electroporation using Neon Transfection System? What parameters and plasmid concentrations that I should use?
Thank you
Hi Lahiru,
Here you have the parameters to perform high efficient electroporation in mESCs using Neon transfection system:
https://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/cell-culture/neon-protocols.par.27415.file.dat/mouse%20embryonic%20stem%20cells-blastocyst.pdf
Regarding the plasmid concentration, it would vary a bit depending the concentration of cells you are planning to transfect and the type of plate/dish. What it's usually recommended is to start with high quality DNA at a concentration of 1-5 μg/μL in deionized water or TE buffer and then adjust it to your format of interest (e.g. 6-well plate). You can use the Table on page 26 of the following protocol as a reference.
https://tools.thermofisher.com/content/sfs/manuals/neon_device_man.pdf
Hope it helps!
Best,
Ruben
Hi Lahiru,
Here you have the parameters to perform high efficient electroporation in mESCs using Neon transfection system:
https://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/cell-culture/neon-protocols.par.27415.file.dat/mouse%20embryonic%20stem%20cells-blastocyst.pdf
Regarding the plasmid concentration, it would vary a bit depending the concentration of cells you are planning to transfect and the type of plate/dish. What it's usually recommended is to start with high quality DNA at a concentration of 1-5 μg/μL in deionized water or TE buffer and then adjust it to your format of interest (e.g. 6-well plate). You can use the Table on page 26 of the following protocol as a reference.
https://tools.thermofisher.com/content/sfs/manuals/neon_device_man.pdf
Hope it helps!
Best,
Ruben
Electroporation might not be the best way to go about transfecting stem cells - they do tend to be sensitive, and could respond poorly to damaging stimuli (such as electrical impulses). Lipofection could be a comparative approach to try (see https://altogen.com/product/mef-transfection-reagent-mouse-fibroblast-cells/) and it should help you avoid any excess cytotoxicity that could arise. You can find some sample concentrations in the protocols for various kits.
Hi Ruben Sebastian-Perez
We did as mentioned in the protocol, but we get around 10-20% transfection efficiency, not 80% as mentioned in the protocol. Is it normal to have this efficiency? or have we done something wrong?
Thanks :)
Hello
Kindly look at links here
Article Electroporation of Murine Embryonic Stem Cells: A Step-by-Step Guide
https://www.thermofisher.com/sa/en/home/life-science/cell-culture/transfection/neon-transfection-system/neon-transfection-system-cell-line-data.html
Hi Lahiru Chamara Weerasinghe Arachchige
Mouse ES cells usually do not present major difficulties in transfection compared with other cell lines. At least based on my experience with mESCs, you can easily achieve high efficiencies after nucleofection (>70-80%).
I would recommend you to include some positive control plasmid carrying some fluorescent protein (if you're not doing it already). In this way, you can monitor by microscopy/FACS if you have achieved the desired efficiency. Some kits already provide plasmids to use as positive controls, as the pmaxGFP plasmid (Amaxa nucleofection kit). Also, it is important to consider that the electroporation/nucleofection systems offer different programs depending on viability/efficiency parameters. If you're interested in high efficiencies because of the nature of your experiment, you should optimize it for this purpose (although you may have little viability). But consider as well that you can purify and enrich the population that has been successfully transfected, if your experimental setting allows you to do it (e.g FACS sorting). Finally, please check the integrity of your plasmid of interest. Run it on a gel (also try to digest it) and re-prep it to ensure that you have a pure and high quality DNA material to work with.
Cheers,
Ruben
Hi Ruben Sebastian-Perez
Thanks for sharing your experience.
We are actually optimizing electroporation parameters for pEGFP, before going to the actual experiment. I do really agree with you that the plasmid purity is highly important for electroporation. Just using milliQ water, instead of EB buffer, we could achieve higher efficiency than before. I will definitely try your suggestions and see how it works
Hi Ted Bremmer
Thank you very much for your suggestion.
Won't it promote the differentiation of ES cells?
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