From: Nick Holmes (nh106@cam.ac.uk)
Date: Tue Nov 18 2003 - 13:47:05 EST
In <URL:news:local.facs> on Fri 14 Nov, Howard Shapiro wrote:
> Stephen Sullivan wrote:
>
> >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. .
> >Does anyone have an
> >alternative way to sort large numbers of heterokaryons from cell hybrids ?
> >Thanks, Stephen
>
> I wouldn't FRET about this...but, all kidding aside, I think there may be a
> somewhat simpler way to identify the hybrids. Michnick and his coworkers
> detect interactions of proteins ("A" and "B" by introducing sequences
> coding for complementary fragments of enzymes such as dihydrofolate
> reductase (DHFR) and beta-lactamase to the sequences coding for the
> proteins under study; when DHFR is used, the interaction is detected by
> binding of fluorescently labeled methotrexate. The heterokaryons should not
> bind the labeled compound, whereas the hybrids should.
I cant see how your suggestion would work Howard (or Stephen's).
The 2 complementary fragments are synthesized on ribosomes in the cytoplasm.
Thus fuse the 2 cells and you can form hybrids. Even if the proteins were
really localised tightly to the nucleus (and I dont think they generally
are), there would be crossover of the newly synthesized proteins after
cytoplasmic fusion, so that the nuclear protein makeup will become
homogenised over time (rate depending on turn over etc.).
I cant think of a reliable way to do what Stephen wants. To really
discriminate between binucleate and mononucleate fused cells seems to me to
require detection of a hybrid structure where the subunits cant mix between
nuclei in the same cytoplasm.
I have a different query tho. Stephen's question implies that when 2
cells fuse at some frequency less than 100% but more than 0%, the nuclei go
on to fuse. Do the nuclei in fact fuse in heterokaryons or does the
tetraploid (subsequently aneuploid usually) nucleus only form after mitosis?
I am sure this question was answered by someone, probably about 40 years
ago.
Incidentally what species are you working in Stephen? I know tetraploids are
stable in some species.
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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