From: Nick Holmes (nh106@cam.ac.uk)
Date: Mon Nov 24 2003 - 09:41:02 EST
In <URL:news:local.facs> on Thu 20 Nov, S. Sullivan wrote: > Nick, Howard et al., > > I am fusing murine ES cells with somatic cells....on average 10% of the > cells fuse. Using a selection regieme, I know at least 1 cell hybrid is > formed per 1000 heterokaryons. > > Nick's question regarding the formation of cell hybrids from > heterokaryons....the heterokaryons need to undergo a mitotic event to form > hybrids...DNA replicates, the nuclear membrane is broken down and the > chromosomes divided and two tetraplod hybrid cells are formed from the > heterokaryon. The cell hybrids are genotypically stable (tetraploid) over > many passages. That is interesting. I alweays suspected that the hybrid nuclei required mitosis, is there hard experimental evidence? Re the stablility though, the hybrids formed by mouse myelomas with mouse lymphocytes are notoriously karyotypically unstable. I also have some experience of karyotypic instability in ES cells. > I am not concerned with the nuclear protein make up becoming homogenised > over time as I will be sorting very shortly after fusion. This puzzles me a bit. By your earlier statement there will be no hybrids until the heterokaryons have undergone S phase and G2/M. How quickly will that happen? > So when I am looking for hybrid cells, I will have an excess of > heterokaryons....using nuclear specificdyes and confocal microscopy would > take me forever. > > Please note that the system does not have to sort hybrids from > heterokaryons with 100% efficiency. Even if I could reduce the > heterokaryon:cell hybrid ratio by a factor of 10 this would be very useful. Given your earlier requirements it occurs to me that you might be able to make do with a very simple 2 colour labelling of cytoplasmic proteins. In theory if you label cell A with, say, CFSE (green) and cell B with CMAC (blue) the heterokaryons (green and blue) could be distinguished from the hybrids by intensity of both green and blue (theoretically half intensity of both). Whether this can be made to work in practise depends on the CV of your intial labelled populations. Obviously the variance needs to be low otherwise you have a significant fraction of cells in your original population that would have half-median fluorescence (and thus heterokaryons of which look like hybrids). The method (in single colour) is widely used in immunology for distinguishing daughter cells after mitosis from undivided parents. However in my experience many cell lines have too wide a spread of initial labeling intensity. Best wishes Nick -- Nick Holmes Division of Immunology, Dept of Pathology Cambridge University, Cambridge CB2 1QP, UK Tel:+44 1223 333871 Fax:+44 1223 333875 mailto:nh106@mole.bio.cam.ac.uk http://www.path.cam.ac.uk/~nh106/
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