Would transfection of iPSCs work using this plasmid?
https://www.addgene.org/132776/
We already tried it using electroporation and lipostem but saw no green fluorescence. Could it be possible that the GFP was silenced therefore not expressed or transfection failed completely? Or the promoter is not the best one for iPSC? we will be trying fibroblasts next.
The plasmid looks good. The promoter may be the issue - stem cells are finicky in what they express. Can you use something like oct4 or alpha or beta actin promoter?
Trying fibroblasts will at least tell you if the plasmid is good.
CMV should be less bright than in fibroblasts, but still visible. Silencing does not happen that quickly.
Do you know that your electroporation settings are getting any plasmid into iPSCs? Are the iPSCs even surviving? They are notoriously difficult to transfect. You need good controls to be sure where your bottleneck is. Get some different plasmids, and try everything in HEK293T to compare against.
Thank you, Aidas Nasevicius the promotor is the issue. It seems we do need to use a EF1α promoter for iPSCs
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