I am trying to transfect primary rat hippocampal neurons with two constructs - one encoding a soluble fluorescent protein and the other tagged protein of interest.
In transfected cells I always see protein amounts for the two constructs that seem to be inversely proportional to one another as judge by the fluorescence. For example cells that express 90% of the maximum of the red signal express only 10% of green signal seen in the highest expressing cells. Cells expressing high amounts of the green tagged protein express little of the red protein...and just about every ratio in between. Ideally I would like to have cells expressing a consistent ratio of each of the proteins for analysis.
The two vectors use the same promoters - but have different polyA sequences. Could this be driving this pattern of expression? However I also see these weird ratios of expression when expressing just two soluble fluorescent proteins from two individual but other otherwise identical constructs.
I have adjusted the ratios of the DNA vectors and also the total amount of DNA, but still seem to see the same variable amounts of co-expression.
Has anyone else seem something similar and have any additional suggestions?
Hi Andrew,
instead of trying to optimize the transfection I would recomend you to use a bicistronic approach (since you mentioned that both proteins use the same promoter). You could either use a plasmid with an IRES sequence to produce a bicistronic mRNA with would give you close to equal expression of both proteins or a P2A sequence, which in theory would give you equal expression. Moreover you could use either an AAV or a lentivirus which would give you much higher efficiencies.
Hope this helps,
Ezequiel
Hi Andrew,
instead of trying to optimize the transfection I would recomend you to use a bicistronic approach (since you mentioned that both proteins use the same promoter). You could either use a plasmid with an IRES sequence to produce a bicistronic mRNA with would give you close to equal expression of both proteins or a P2A sequence, which in theory would give you equal expression. Moreover you could use either an AAV or a lentivirus which would give you much higher efficiencies.
Hope this helps,
Ezequiel
Andrew, using two plasmids with the same promoters for transfection isn't recommend, because they compete for transcription factors binding and lead to the distortions you observed. To overcome this problem, using a P2A sequence as suggested above would be the best strategy to have an even expression of both proteins.
Good luck!
Hi everyone, I just want to add another following-up question to this thread. I've been trying to optimize the transfection protocol with Lipofectamine 2000 to express 3 different constructs (2 of them are about 9 kb with CAG promoter, and the third one is about 4.3 kb with CMV promoter) in rat hippocampal neurons. This seems to be very challenging so far, especially because the larger vectors carry two fairly weakly expressed genes (SEP-GluA1 and myc-GluA2). Besides making tricistronic/bicistronic vectors, do you have any other recommendation? I would appreciate any suggestions! Thank you so much! :)
Hi Huong,
the typical things to try would be to modify the DNA/lipofectamine ratio to have the perfect balance between transfection efficiency and low toxicity. If you have a good ratio that works well in your particular experiments I would stick with that.
One important thing to consider, as you suggested, is the size and promoters of the plasmids you are using. Since you didn't give any specifics I don't know how much you are adding of each of them but you should remember that if you are adding the same amount of all three plasmids you would be adding more than double the number of plasmids with the smaller size. You could start with equimolecular number of all three plasmids and then modify from there. You could also try single transfections first to stablish what is the level of expresión you can expect for each individual construct as well as the strength of the promoters in your system; then you can make adjustments to add all three. Finally, I would suggest to mix all three plasmids thoroughly before adding the optimem (or similar); I personally don't think that is critical but I have met many people that swear by it.
Hope this helps,
Ezequiel
Hi Huong,
I have also heard (albeit only anecdotally) that long-term expression of the GluA1-SEP constructs can be quite toxic to hippocampal neurons. I'm not sure at which time point you are transfecting vs utilizing the neurons, but I think many of the published reports using these constructs only overexpress them for a day or two before using the neurons experimentally. So as Ezekiel mentioned, it may be useful to check the health of your neurons with the single transfection of GluA1-SEP or GluA2-myc.
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