I'm working on meiosis in S. cerevisiae. Cells that go through meiosis "become" an ascus with 4 spores (tetrads). Some mutants (spo13 null for example) go through only 1 division in meiosis though, and form asci with 2 spores (dyads).
If I somehow tag individual spores (say the nucleus) with a fluorescent protein, I can easily differentiate between tetrads, dyads, and normal cells as these would have 4, 2 and 1 fluorescent dots, respectively. Is it possible for a FACS machine to sort out only those cells (asci, actually) that have 4 dots?
Or at least, sort out asci with a fluorescence intensity (or amount or whatever, not sure on the terms) of X but not X/2 or X/4? I'm assuming that the nucleus of spores in tetrads isn't smaller than that of spores in dyads or normal cells.
hi
do you need them to be alive when you're done sorting? if not, you can permeabilize them and add a DNA-binding stain (for example propidium iodide) and sort on the amount of stain. I haven't worked with spore tetrads, but in mammalian cells you can certainly sort on 2 fold differences in stain intensity.
if you have a way of adding a flourophore without killing them, then yes, you should also be able to sort those containing four tetrads. give it a try.
I do need them alive, but I could use a GFP tagged DNA binding protein.
In any case though, the problem is that the amount of DNA doesn't differ.
maybe the density of the cells changes in this process and you could use physical methods prior to sorting to separate?
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