transfection of single copy of plasmid per cell - (Feb/15/2005 )

Hello All,


Does anyone know of a transfection procedure where mamallian cells can be transfected and will result in a single copy of the plasmid for each cell transfected?

Something like transformation of E.Coli, where it is thought that only a single copy of the plasmid transforms each cell.

To my knowledge, with the current mammalian transfection techniques many copies of plasmid are transfected into one cell. Or am I wrong?

So, if anyone can give a suggestion how can I transfect mammalian cells with only one plasmid for each cell, I would appreciate it.

Thanks.

Tau

I know nothing about mammalian cell transfection or transformation in mammalian cells in general, but when I think about it, Ive never heard of mammalian cells maintaining plasmids (circular DNA with a selectable marker).

If this is the case, then youll need to make sure your DNA gets into the genome. Remembering way back to college, you might try some sort of "knock-in" strategy via a double crossover using a cassette.

Just a stab in the dark, and Im probably wrong so take my reply with a grain of salt

Good luck

Your knowledge is correct--each cell that actually gets transfected receives a large number of DNA molecules. There is no way currently to make sure that each cell only gets one plasmid. There are two possiblities to approach this goal, but neither is great. Whatever your transfection protocol, the DNA is clustered or attached to something that will carry it into the cell--a small crystal in CaPO4-type transfections or a liposomal-type grouping in transfections using reagents such as Lipofectamine. You could possibly greatly dilute your DNA in an attempt to put just a few molecules of DNA with either the crystal or liposomal carriers, but you would also greatly reduce your transfection efficiency by doing this and you might not be able to find any cells that actually got transfected. Otherwise, cells maintain the plasmids after transfection, but do not usually replicate them (the one exception to this that I know is if you are using HEK293T cells and a plasmid with an SV40 ori), so the plasmids will dilute with each division. They don't necessarily dilute evenly, so you will probably end up with some daughter cells containing the majority of the plasmids. But you could select or sort cells that were transfected (antibiotic or fluorescence marker) and maintain them and see if you got closer to what you want. If what you're actually looking for is lower expression of your transfected protein, you're better off looking at an inducible expression system, in which case you might be able to titrate the drug/chemical used to induce the protein (ex. tetracycline or doxycycline) or trying to change the promotor in your vector to one that is not so potent. Good luck.

Thank you.

Tau