From: S. Sullivan (sjs93@cam.ac.uk)
Date: Thu Nov 13 2003 - 09:41:32 EST
I am studying nuclear reprogramming in cell hybrids. I want develop a FACS based system that will allow me to sort heterokaryons (fused cells where the two diploid nuclei from the fused cells are still distinct) from cell hybrids (where the nuclei have fused to form a single tetraploid nucleus). I am currently thinking about using FRET (fluorescence resonance energy transfer) to allow me so discriminate between heterokaryons and cell hybrids. I plan to label two cell types with either an arbitrary nuclear protein A linked to CFP or another nuclear protein B linked to YFP. The nuclear two proteins will bind specifically and irreversibly to each other when mixed and energy transfer should occur. So in the experiment, one cell type will be labelled with nuclear protein A-CFP and the other cell type with protein B-YFP. I am guessing that in heterokaryons where proteins A & B will be separate and energy transfer between the CFP and YFP will be minimal. In hybrids however, proteins A & B will bind and energy transfer will occur. I am assuming that proteins A and B need to be small and bind specifically to each other to maximise FRET. I have never measured FRET before. Is this system like to work? Does anyone have any suggestions how I might optimise this system. Does anyone have an alternative way to sort large numbers of heterokaryons from cell hybrids ? Thanks, Stephen -- Stephen Sullivan Wellcome Trust/Cancer Research UK Institute Tennis Court Road University of Cambridge Cambridge CB2 1QR, United Kingdom Tel: + 44 1223 334137 Fax: + 44 1223 334089
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