Chromosome staining and counting

Hi Dear Worm Community,

I am working on a mutant with some sterile and large body-size progeny and male appearance in the progeny.

I am assuming that there could be a chromosomal non-disjunction event happening in that mutant.

     I would grateful if anyone can tell me:
  • What kind of staining or method I can use to detect the extra chromosomes or loss of a particular chromosome.

  • What cells are the most suitable cells to count the number of chromosomes?

Best Regards

you can do some live staining with hoechst. Try a range of concentrations, and incubate worms 2-3h with some food, then look under the scope with a DAPI filter. Of course, you need to find cells in metaphase, and reasonably large nuclei, so it limits you to hypodermis (if you catch them between larval stages) or gonad (the gut might also be a possible target. There’s some endoreplication, IIRC, but then again it would make counting chromosomes extra-tricky).
Another method, my favorite, is to go for a histone::GFP protein fusion. ppie-2::HIS::GFP works fine (reasonably strong staining in the oocytes, allowing you to count chromosomes as they segregate during meiosis and the first few stages of embryogenesis. There must be at least one strain at the CGC). You’d need good eyes and/or a nice confocal, though, but I’ve done it before with just an epifluorescence scope at 400x.

Since you have sterility and males, it seems that you are effecting meiotic chromosome segregation. How high is the percentage of males and is there embryonic lethality? I would start by looking at the germ line and seeing if there are any obvious cytological defects. You can dissect out the germ line, get it to stick to poly-L-lysine coated slides and mount them in Vectashield with DAPI. You should see six DAPI stained synapsed pairs of homologs at the end of meiotic prophase I, towards the end of the germ line. More than six would indicate issues in synapsis, pairing, break formation etc. Fewer than six would indicate aggregation due to aberrant repair of meiotic double strand breaks.