Hi All,
Recently I tried to make a GFP-ES cell line which can express GFP strongly. I used pCDH Vector(PKG-GFP) and packaging lentiVirus to transduce my ES cell. However, the GFP is so weak so I can hardly see it when it's signal. Well as ES cell will form cluster, I can clearly see GFP when many ES cell come together.
But the problem is I need to induced them become neurons, and try to do a co-culture assay, so I need to distinguish them with another cell (no GFP), and I can't see them by eye when it form signal. And when I use AntiGFP Ab, it seems has strong background.
Anyone has any ideas? If I want to change the vector for it, will CAG be a good promoter?
The brightness at the ESC stage might not necessarily predictive of what's going to happen in your differentiated neurons. I think it may be useful to actually take them to the neuron stage and re-assess what happens to the promoters and brightness level then.
The promoter intensities should change as the cell differentiates... PKG I think should be weak-ish in either state. CAG I think will look weak at first, at the ESC stage, but should gain brightness as it becomes a neuron. (assuming your differentiation protocol is complete enough to actually accomplish all the gene expression changes involved in that). I haven't seen it myself, but heard good things about EIFa promoter at the ESC stage.
Also, a correctly differentiated neuron should stop dividing, and thereby get more time to accumulate intracellular GFP protein, before diluting it by cell division. So it might be substantially brighter just for that reason.
nowadays, I use CAG promoter from addgene 20071, it seems great at begainning, strong GFP in ES cell, then when it become neuron, no GFP anymore....
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