After infecting pancreatic beta-cells with two adenoviruses encoding two different fluorescent proteins under the CMV promoter, it seems that cells express more intensely one or the other but rarely both proteins at similar levels. The same is true when one of the protein is under control of the rat insulin promoter. Do you have any idea why and how to solve this problem? We already tried to infect them with both viruses at the same time, but it does not help.
Hi Jean-Christophe,
You raise an interesting question that may have different answers depending on the precise example. At the transcription level different cis-acting elements (e.g. part of the protein coding region of the fluorescent protein) could influence the strength of the CMV promoter. Even when transcription is comparable, the protein half life could be very different. At a more proximal step of the experimental operation the efficacy of the adenovirus system to get information into the nucleus may vary. For each of these causes for differences cell-type specific mechanisms could be responsible.
best regards,
Frans
Hello Jean-Christophe, little to add to what Frans already said. Anyway, just a few considerations.
1. How are you estimating the protein levels? In our experience, in Western Blotting fusion proteins often alter (and reduce) the affinity of an antibody for the non fused protein (sometimes actually the Ab becomes totally ineffective), so to quantify your protein levels by WB you should calibrate your Ab with the in vitro synthesized or bacterial/yeast expressed fusion proteins. If you quantify by fluorescence, again, different fluorophores have different quantum efficiency, not to talk of the detectors variability, so you should again calibrate (which might be very difficult). Finally, have you checked the mRNA levels (qPCR)? At least you would learn at which level is the problem (RNA transcription/stability or protein translation/half-time)...
2. If the final expression of your fluorescent proteins is not intrinsic to the nucleotide sequences or the proteins themselves (as pointed out, transcription or protein stability might be different) you could clone the two proteins in the same adenoviral vector, unless they are very large they could fit (you have in total about 8 kb of space).
Good luck!
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