Dear Flowers, I have been looking at promoter function in transduced cells using a GFP reporter for some time and am generally unhappy with the rigour of the 'answers' I am getting. What I really require is a statistical method which gives me a reproducible constant. The problem is that the 'true' level of expression should be the fluorescence I would get off of 1 copy of integrated genome (inc promoter under investigation and GFP cassette) per cell. However, the number of copies per cell will vary (normally) I suppose, and also as a result of my transduction efficiency. As my transduction efficiency improves I would suppose hypothetically that the modal number of copies per cell would increase. Anyway at present I am assuming that at a low transduction percentage any cells transduced will likely only include (and express) 1 copy of GFP. Therefore, after removing non-transduced cells, the geometric mean of those remaining should give me the expression level of GFP, which when normalised against a control vector gives me a benchmark. However, having reviewed my accumulated data it is often difficult to see any constant expression at lower levels of transduction. Also the means by which I remove the transduced from the non-tranduced cells is problematic. At low transduction levels where expression may be fairly low it is often the case that the transduced population is merely a 'shoulder' on the the untransduced population when viewed on a 1 parameter histogram. I have found no 'satisfactory' way of determining transduction percentages in these cases. It has occurred to me that at at transduction levels approaching 100% the geometic mean might give you an answer that is constant and comparable between vectors. Might this not be a better approach? (it would use up a lot of raw materials though). Hoping someone out there understands what I talking about, and can give me a few hints Daniel Chipchase Research Assistant
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