We are planning to over express a particular protein in endothelial cells. Would like to hear comments from an experienced people with mRNA transfection kits and conditions .
Hello,
Handling a plasmid is easiest than mRNA and permits to select clones as it's integrated into the genome. But to produce a protein, mRNA is safer, faster and much more productive. It will directly instruct the cells once delivered into the cytosol (as early as 2-4h) while pDNA needs first to reach the nucleus, be transcripted etc
Working with mRNA requires using modified mRNA (capped and polyadenyled) but there are commercial kits doing that. You need to produce a stock only once, make aliquots and store them in the freezer. Use them whenever you need to make transfections.
another option is simply to buy your mRNA to a supplier like TriLink, US.
For more info, you can have a look to what we achieve with our Viromer RED for example
https://viromer-transfection.com/mrna-transfection
Hope that helps!
Bests, Sandra
mRNA transfections are suitable in systems where you need a short term expression of a protein of interest (e.g. imaging surface expression etc). It is routinely done in oocyte systems. Needless to say, mRNAs do not last for too long intracellularly, so time would be a limiting factor.
Plasmid DNA transfections or lentiviral vector-based gene expression is more long-term and if you use inducible systems, you can turn it on/ off depending on your experimental needs. Selection markers like puromycin resistance will ensure that the DNA expression vector is still present in your cell type.
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