Transient expression of a4b2 neuronal nicotinic Ach receptors in HEK 293 cells.
We tried to transiently transfect HeK Cells with neuronal a4b2 nachr+CFP, but found very low expression. When we tried to patch we found receptor is not responding at all.
Can anybody explain why the expression is so low or is this the trend with transient transfection? Also, what should be the amount of plasmid that must be taken for transfection to have good result in HEK 293? Is permanent cell line the only solution?
Hi Abhilasha,
If you can provide some more information on your protocol, hopefully we will be able to help you. Here are some questions that would help us understand your problem:
1. What reagent are you using for your transient transfection?
2. Can you please describe your transfection protocol in detail? How much transfection reagent, how much DNA, how many cells, what size plate/well, how long do you leave the transfection reagent on, do you change media before/during/after transfection, how long do you wait to check for expression...?
3. Are you transfecting with one plasmid that has both a4b2 nAChR and CFP together (bicistronic), or are these 2 separate plasmids that you are pooling together into one transfection?
4. What are your controls?
5. How did you determine your expression levels (Western blot, clamping, CFP fluorescence, etc)?
6. Can you transfect these same HEK cells with something else that works well?
In general, transient transfection can be very effective -- after optimization. There are lots of variables that can affect transfection efficiency:
A. Cell density
B. Cell health
C. Transfection reagent
D. Ratio of transfection reagent to DNA
E. Transfection protocol
F. Quality of DNA for transfection
G. Mycoplasma contamination
... and the list goes on. Hopefully, once you provide some more details we will be able to determine which variables are most likely to be at work in your case.
Best,
Irit
Hello Irit,
First of all thank you for giving your precious time and long reply. The answers of your questions are given below:
1) We use Calcium Phoshpate method for transfection.
2) We use 2.2 μl of mix (plasmid+CFP). Therefore,total DNA in 2.2μl is 3 μg.
Transfection reagent : Water+Calcium+BBS= 327.8μl.
We use 24 well plate but we do not count no. of cells before plating.
Actually our transfection procedure is of 3 days. First day, we plate cells in a petri dish (23 cm2 area) containing 3ml of HEK medium. Next day the cell are almost 60-70% confluent. Then we change the medium and incubate it for one hour at 37 degrees. After that we mix transfection reagent and plasmid and incubate it for 10 minutes and add it to petri dish. Then, next day we plate the cells in 24 well plates contining the coverslips and fresh medium. After that we wait for 5-6 hours and then, we patch.
3) No, we have different plasmids for a4, b2 and CFP.
4)I didn't get your question
5) CFP fluoroscence and clamping.
6) Yes, when these HEK cells are transfected with muscle nachr (same procedure of transfection) , they work well and we have good results too.
I hope we are doing in a right way.
Hello Abhilasha,
I have transfected mammalian cells, including HEK cells, with the beta2-CFP nAChR construct and other FP-labeled nAChR subunits. I used several popular commercial products for transfection. The one with the best yield for transient transfections was ExpressFect by Denville Scientific. We used the manufacturers directions with two modifications (1) we only incubated for 3 hours at 37 degrees and (2) we did not use any antibiotics in the media. These modifications greatly improved cell viability while maintaining the normal transfection rate.
As a side note, I had no problems with alpha4-YFP expression in HEK cells. However, I initially had a great deal of difficulty with alpha7 expression in HEK cells and also had lower expression levels in other cell lines. That was not a problem after co-transfection with ric-3.
These steps and other details are published in TA Murray, Q Liu, P Whiteaker, J Wu and RJ Lukas (2009) Nicotinic acetylcholine receptor alpha7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors. Acta Pharmacologia Sinica 30 (6): 828-841; and TA Murray, D Bertrand, RL Papke, AA George, R Pantoja, R Srinivasan, Q Liu, J Wu, P Whiteaker, HA Lester, RJ Lukas (2012) alpha7beta2 nAChRs assemble and function, and are activated primarily via their alpha7-alpha7 interfaces. Mol Pharmacol, Vol. 81(2): 175-188.
I wish you success.
Teresa A. Murray
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