I am trying to transfect HeLa cells using three different plasmids. These three plasmids are of the same vector (CMV2-flag inserted at the N terminus) but a different construct of the same protein was inserted the same way (protein mapping). The plasmids' sequences have been verified by sequencing analysis. Additionally the purity of the plasmids is very good according to nanodrop 260/280 and 260/230 analogies as nanodrop states. The Transfection method i use is the calcium phosphate protocol (Sambrook). The problem i face is that the transfection efficiency varies between the three plasmids and the expression of the constructs as well. Cells have been stained by a-flag and seen under IF. The first has a transfection efficiency 30-40% the second 5-10% and the third 0%. The first has a low protein expression the second has a very intence expression and the third one no expression at all (probably the plasmid i have already isolate a new . Analysis by electrophoresis and WB, shows a very clear band for the second one (5-10%), and a really faint band for the first one (30-40%).
What can i do to maximize the transfection efficiency where needed and more importantly why there is such a difference in expression levels.
thanks
Giannis
I have not worked with these cell lines, but if transfection efficiency is low, you can try the following:
1. test a (wider) range of different ratios of transfection reagent and plasmid / DNA concentrations
2. try a different transfection reagent (Lipofectamin and Polyfect work well, though yes, calcium phosphate might be a cheap and effective alternative)
3. If your target construct is rather large, you can try smaller control vectors to exclude that vector size is an issue.
4. Some people use chloroquin to enhance transfection efficiency.
5. Although it shouldn't be an issue, but is sometimes missed for exactly that reason, you should (re-)check that your promoter is working in the cells you are using. If those cells are commonly used by other researchers as documented by the literature, check whether they do anything different and try their protocols.
6. Check for mycoplasms in your cells. Contamination can really decrease transfection and transduction efficiency.
Furthermore, if your low transfection rate is not only in control cells, it might be possible that there is a biological effect. The expressed construct might be targeted for degradation by intracellular pathways, which you did not know of beforehand. In that case, you could check for mRNA levels. Sometimes, construct effects might also cause cells to throw out the plasmid again.
If your lab is equipped for that, you might also want to consider viral transduction instead of transfection. Usually, this is quite efficient.
Finally and if nothing helps, whether you really need a high transfection efficiency might also depend on the experiments to be used. For subsequent Western blotting, higher transfection rates are often needed to clearly make out biological effects. However, immunocytochemistry, for instance, also allows you to work with lower transfection efficiencies as long as you are capable of identifying transfected cells (e. g. by having flourescence tags). If you really need a specific cell line, which does not work well in transfection, you might consider using ICC instead of Western blotting, although there are, of course, some disadvantages to that.
Hi
I worked with HeLa cells in the past and theses cells are usually very easily transfected. I’ve used electrophoresis and Jet Pie or Jet Prime. If you are using the same expression vector and the only difference is in the cDNA, then I would assume that the cDNA is responsible to the differences in the expression levels.
So:
1. Try using a specific Ab. Targeted against the expressed protein rather than the Flag-tag – maybe there is some processing at the protein level and the N-terminus is cleaved.
2. Try and examine the expression levels at different time periods: 24, 48, 72 hr.
3. Try and examine the cells viability – maybe the expressed protein is toxic.
4. And finally, try to block proteosome degradation by adding MG132 to the cells – maybe the protein is send to degradation.
Good Luck
Rona
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