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Since we provide both constitutive and tetracycline-inducible versions of both U6 and H1 shRNA promoters, we often get questions regarding which to choose or which is “better.” In fact, our data with both promoters indicates they function equally well, so it is difficult to provide a clear-cut answer. We use the U6 promoter primarily for shRNA expression libraries as this is generally thought to be a stronger promoter in most cells. We have not systematically tested this in-house, but it is consistent with our findings where U6-driven constructs provide knockdown in a wide range of cell lines. However, some of our clients have specifically requested H1 constructs because U6 constructs appeared detrimental to the growth of some cell lines, possibly because expression levels are too high. The particular concern mainly seems to be with using U6 in neuronal cells. Just to emphasize, we have not seen this in our research. Conversely, we have not had problems with H1-driven shRNA constructs. In a wide range of cells, it seems to function as well as U6.

Overall, we don’t have a strong recommendation regarding the use of either promoter. It may be that one or the other is better in certain cells or under certain situations. However, in over 50 cell lines that we have worked with, we haven’t seen a clear distinction. We do suggest the researcher try both in their cell systems to check first-hand if one is preferable over the other.

We would be interested to hear from anyone who has had experience with U6 and H1 promoters and what their results were. Comments can be posted in the form below.