Hi,
I want to transfect hiPSC-derived neural stem cells (Axol Bioscience) with DREADDs plasmids (pAAV- CaMKIIa-hM3D Gq, Addgene) using the Lonza 4D Nucleofector system. The protocol I am using works well when I do the electroporation with pmaxGFP, however, not with the DREADD plasmid. At this point, my main issue is that there is no expression of the DREADDs in the nucelofected cells even after 48hrs. But I see GFP expression in the control so at least, I know some parts of the protocol works. I suspect maybe the DREADDs plasmid is too large?
I've even tried a few different pulses, (CB-150, DS-112, DS-113) and I was wondering if anyone has some recommendations. If the plasmids are too big, how can I best troubleshoot this problem?
The concentrations I use are:
P3/P4 nucelofector solution (82ul)
Supplement 1 (18 ul)
Plasmid/GFP (2ul)
Thanks for any help!
I have a couple of questions first:
-Does the DREADDs plasmid you're using have a reporter? I see a few listed that have mCherry fusions. If you don't have a reporter, how are you measuring transfection efficiency (as opposed to efficiency of transgene expression)?
-Are you certain the CaMKIIa promoter expresses well in NPCs?
-Have you tried transfecting a more mature neural cell line to ensure your plasmid works as expected? Often small changes in plasmid sequence can lead to loss of expression/plasmid stability.
I don't think plasmid size should be an issue if your construct is less than 10kb. Experiments to answer the above questions will probably help solve your problem.
Hi, thanks for the response. The protein has mCherry fusion. As for the CaMKIIa, I am not completely sure, but is a great point that I have already been checking.
I can try with another cell line to test the efficiency of the plasmid also. For now, I am checking to see if it is a matter of letting my cultures reach maturity. Previously, I've been checking for expression after 48hours, then deeming it a failure. I will try to keep them longer and see.
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