I’m currently trying to transfect LAN5 cells (human neuroblastoma cell line) using the Lipofectamine 3000 transfection reagent, both with the vector pCMV6-AC-GFP expressing the human heat shock 60kDa protein (Origene RG224428 vector) and with a control plasmid (the same vector without the insert, Origene PS101000 vector).
Using a DNA : Lipo ratio of 1:3, I obtained a good (but not high) transfection efficiency with the control plasmid, but no transfection, or a very low transfection efficiency with the expression plasmid.
Could the plasmid size be responsible for this different transfection efficiency?
Does anyone have any advice to improve the transfection efficiency for both plasmids?
Thanks for your suggestions!!!
I found that the quality of the plasmid matters more than the size. If some portion of the plasmid is present in linear form the transfection efficiently would be lower. The best option is to have supercoiled and circular forms of the plasmid
True. If your plasmid size is big, it would make hard sometimes for transfection. To improve your transfection efficiency, you may use a transfection reagent supplemented with a condenser.
The topology (linear or supercoiled) and the size of the vector construct, the quality of the plasmid DNA, and the promoter choice are major factors that influence the efficiency of plasmid DNA transfection.
Some useful guidelines can be found at https://www.thermofisher.com/fi/en/home/references/gibco-cell-culture-basics/transfection-basics/guidelines-for-plasmid-dna-transfection.html
Hi Alessandra
It is generally true that a bigger plasmids are difficult to transfect than smallers ones, but take into account that you have a 6.6kb plasmid that is transfected well (more or less) and a 8.3Kb with low or no transfection at all. 1.6Kb doesn't seem to be such a difference to justify that, but well...could be.
For starters, you can't blame neither L3k nor the cells or your hands, because the control is working. So it has to be the plasmid. You can't do anything about the size, but you can ride out other things, some suggestions:
1) Repeat the preps, sometimes DNA got contaminated with endotoxins or whatever E.coli material, quality of the DNA is even more critical than size in some cell lines, as pointed out by Arpine Sokratian and others previously.
2) Check your plasmid and control plasmid along in an Agarose gel BOTH digested and uncut. Sometimes, E.coli likes to mess with origin of replication and the plasmid can be a dimer (much bigger). This can be checked if your uncut plasmid run way higher than expected in the gel when compared to the control.(Normally supercoiled plasmid should run faster)
3) How are you checking transfection efficiency? In your control , eGFP is expressed alone, but in your plasmid, eGFP is expressed as a fusion protein with Hsp60, so the expression levels could be affected by the fusion or the effects of this protein in the cells. Thus, fluorescence could be lower, making more difficult to identify low producer cells through a microscope. In this case, efficiency should be assesed by FACS comparing cells transfected with the control plasmid, with the problem plasmid and untransfected. Untransfected cells will mark the threeshold in order to consider wether a cell is positive or not.
4) Of course, you can test other reagents(i.e. https://www.biontex.com/en/k4-transfection-system.html) or protocols (i.e slow centrifugation of cells and polyplexes in order to enhance efficiency)
i hope you can find something useful for your problems. And, if you please and she's still running there, send my best regards to Federica Scalia
Best of Luck!!
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