Yes presently I am transferring transduced cells (some expressing GFP) to 32 degree incubator for 3-4 days after an intial period of 2 days at 37C post transduction. Cells are then trypsinised and FACSed with TO-PRO-3 used as a dead cell discriminator. PS maybe I didnt make it clear I was transducing cells with a viral vector encoding a GFP expression casette, and was looking at low transduction %ages on the assumption that these would most likely be single 'hits'. I wondered whether anyone out there had done similar work or had some rigourous methodology for gauging 'true' expression levels as I couldn't find anything in the literature (plenty on transduction, very little on expression). -----Original Message----- From: Dennis J. Young [mailto:djyoung@ucsd.edu] Sent: 22 January 2002 18:18 To: cyto-inbox Subject: Re: Statistical methods for Expression Analysis While GFP is easy to stuff into cells, it needs to be expressed at hundreds to thousands of molecules pre cell to be detected. It also needs to mature in order to become fluorescent. And, lastly, it stays around for days before breaking down! At 04:23 PM 1/17/2002 +0000, you wrote: >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 ---Dennis Dennis J. Young Flow Cytometry Core Facility University of California, San Diego Internal Medicine Group, Bldg #4, Room 126 9500 Gilman Drive La Jolla CA 92093-0671 Mail:<<mailto:djyoung@ucsd.edu>> WWW:<<http://cancer.ucsd.edu/flow/>> Telephone:(858) 822-0407 FAX: (858) 822-0403
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