Nature Methods | Methagora

Idiosyncrasies of the human CMV promoter

A Correspondence in the May issue of Nature Methods points to the fact that the widely used human cytomegalovirus promoter shows dramatic differences of expression in various cell types.

The Correspondence focuses on potential problems associated with CMV in the brain in relation to a paper by Wickersham and Callaway who respond to the criticism.

Share your experience with CMV in neurons or other cell types by adding your voice to the discussion.

Comments

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    Martin Hafner said:

    I used a CMV-ßGal vector to normalize expression of luciferase reporter gene constructs back in the 90s. Unfortunately, CMV-ßGal expression was quite variable in unrelated experiments. Finally we replaced CMV-ßGal by an EF1-alpha-ßGal construct which gave similar results in independent experiments. A colleague told me that they also had problems when they tried to use the CMV promoter to drive the expression of resistance genes in targeting vectors and they’ve shifted to the human and murine PGK promoters .

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    Damian G. Wheeler said:

    I showed in a 2001 paper in JBC (276:31978-85) that the CMV promoter functions poorly in cultured hippocampal and sympathetic neurons, whether the promoter is transduced via adenoviral transfer or is in the context of chromatin. Interestingly, we found that the CMV promoter can be strongly induced in neurons by stimuli, such as neuronal activity, that activate the transcription factor CREB: this is mediated via 5 CREB binding sites in the promoter. Often, people tell me they have good success with CMV when they transfect neurons with traditional techniques (lipofectamine, Ca2+ phosphate); however, this likely stems from activation of the promoter when the cell membrane is disrupted during transfection. Indeed, if one depolarizes hippocampal cultures days after transfection, they will induce expression from the CMV promoter in many cells that otherwise would appear non-transfected, revealing that the transfection efficiency is much higher than thought. Also, CMV might function well when delivered by certain viruses due to cross-talk from viral genes activating CMV. Finally, we showed in a subsequent paper (Mol Cell Neurosci. (2004)26(1):50-62) that ongoing synaptic activity in culture produces robust expression from CMV through the CREB binding sites. Thus, expression seen in neurons in vivo may be related to the level of activity, and not simply whether or not a particular neuron is infected. The bottom line is that, under most circumstances, the CMV promoter should be avoided when working with neurons, something that is, unfortunately, often not recognised.

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    Adalberto Merighi said:

    I agree with the observation that expression of the hCMV changes dramatically among cell types. We have used the pEYFPN1 to drive expression of BCL2 into cerebellar neurons after biolistic transfection of postnatal (P8) slices of mouse cerebellum. Only the cerebellar granule cells were successfully transfected with no transfection of other neurons and glial cells.

    The observation of Wheeler and colleagues and his comment above point out that expression of hCMV is induced by neuronal activity. However when we have compared the levels of expression of pEYFPN1 (the empty vector) to those of p-EYFP-hBCL2-N1 we have observed that whereas expression of the empty vector is not affected by 25 Mm KCl, expression of the fusion protein is increased by nine fold.

    Thus it is possible that regulation of hCMV is more complex that speculated and that studies in vivo (in such as those in transgenic animals)and/or ex vivo (in slices) add further complexity compared to those in cultured isolated neurons