Hello~ I am trying to transfect GFP-N1-5-HT1a into N2a cells using lipofectamine 3000. Before, I tried GFP-C1-5-HT1a with the same experimental condition, and the transfection efficiency can be 50%. But, now, for GFP-N1-5-HT1a construct, the transfection efficiency was only 20%. Is there any problem with the fusion of two fragments of DNA or transformation? Or is there any method or reagent that I can use for DNA with bigger molecular weight?
Yes, higher molecular weights do impede transfection. There are two things you can do to improve the overall results; use a complex condenser, and use a transfection reagent pre-optimized for N2a cells (https://altogen.com/product/neuro-2a-transfection-reagent-neuroblastoma-cells-ccl131/). The complex condenser will compact down your liposomal complexes for cellular entry and that should give you the higher efficiency needed for most types of research.
Hi Mengya,
Transfection of larger plasmids is less efficient and I hope someone has a suggestion.
Concerning your constructs: I guess they differ by the position of GFP. Can differences in the Kozak sequence be one of your problems? In this case you may not detect cells with low expression. On the other hand the N-terminal GFP may give better results even if the fusion protein is not properly expressed. To check this I recommend a Western blot with an antibody directed against GFP.
Have you tried using PEI (polyethylemeimine)? Both linear & branched PEI(s) have significantly better transfection efficiencies compared with Lipofectamine. In our lab, we have successfully co-transfected upto 12 different plasmids (combined Mw > 70 kDa) using PEI.
From what I have heard from my lab mates , n2a cells don't "lipofect" well. I have used nucleofection with really good efficiency in n2a cells.
This might not be the case, but just if you have time/resources, you can do an experimental design and determinate which factors increases the yield of your transformation. Factors as concentration, time, temperature, volume, might improve your transformation. If you succeed, your future colleagues will have an established protocol with a great yield! :)
Hi Mengya,
I can also recommend another technology called Magnetofection which has been successful to transfect difficult cells with large DNA
http://www.ozbiosciences.com/content/13-magnetofection
For your application, PolyMag Neo or Magnetofectamine reagent are good options
Good Luck
Olivier
Yes, higher molecular weights do impede transfection. There are two things you can do to improve the overall results; use a complex condenser, and use a transfection reagent pre-optimized for N2a cells (https://altogen.com/product/neuro-2a-transfection-reagent-neuroblastoma-cells-ccl131/). The complex condenser will compact down your liposomal complexes for cellular entry and that should give you the higher efficiency needed for most types of research.
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