From: Howard Shapiro (hms@shapirolab.com)
Date: Thu Nov 13 2003 - 20:39:36 EST
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. 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
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.
Remy I, Michnick SW: Clonal selection and in vivo quantitation of protein
interactions with protein-fragment complementation assays. Proc Natl Acad
Sci U S A 96:5394-9, 1999
Remy I, Wilson IA, Michnick SW: Erythropoietin receptor activation by a
ligand-induced conformation change. Science 283:990-3, 1999
Galarneau A, Primeau M, Trudeau LE, Michnick SW: Beta-lactamase protein
fragment complementation assays as in vivo and in vitro sensors of protein
protein interactions. Nat Biotechnol 20:619-22, 2002
You would be able to discriminate both heterokaryons and hybrids from
single cells by DNA content using Hoechst 33342 (or possibly DRAQ5, if the
cells will survive exposure to it), or you could use two tracking dyes,
e.g., PKH26 and one of Molecular Probes' red-excited long side chain
cyanine derivatives, and pick out heterokaryons and hybrids based on dual
staining. I doubt that this will be harder than measuring energy transfer.
-Howard
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