Hello - Can anyone give me insight as to potential markers that could be used to bind and mark the cytoskeleton of mouse oocytes? However, I would want to use these in live oocytes (devoid of their ZP), so the marker/fluorophore should be cell permeable. Is this feasible?
Thank you very much in advance.
Hi Sylvia,
For visualization of cell compartments in live oocytes, people usually inject mRNA encoding fluorescent markers. They leave zona pellucida (as it keeps the cells more resistant) and this way you also avoid using any drugs.
Usually you isolate the oocytes from the ovary in medium containing dbcAMP (to prevent them from resuming meiosis as after that happens they express exogenous RNA at much lower levels). Then you onject them with your mRNAs, incubate for about 3-4h , release in medium without dbcAMP and image (or wait until they reach the stage you are interested in, for example metaphase II).
Good cytoskeleton markers are then fluorescent versions of:
- alpha-tubulin (gets incorporated into microtubules)
- MAP4 (interacts with existing microtubules, gives a nice signal with less of background, yet if overexpressed might lead to unwanted stabilization of microtubules)
- utrofin for actin cytoskeleton
- phalloidin (but his one is a bit tricky because it really does stablize actin cytoskeleton, which might lead to unwanted effects on the cell).
I haven't met anyone doing it differently in live oocytes.
Best wishes,
Michal
Thank you for your helpful answer, Michal. Unfortunately microinjection is not a possibility for this particular project as I will be handling several 100s of oocytes for each experiment. The point is to use something membrane permeable. Eventually these oocytes will be lysed and we want something that allows us to follow the lysate. However, we cannot stain the lysate either. It is complicated. Any ideas? Is utrophin cell permeable?
Hi Sylvia,
I agree, injecting several hundreds of them doesn't sound feasible!
Unfortunately, utrophin is a protein and will not go through membranes. Phalloidin does not penetrate well, but there are modified derivatives that can be used easier in live cells. I think Merck Millipore sells some. Also check out his paper for more info about probes for cytoskeleton in live cells:
http://www.nature.com/nmeth/journal/v11/n7/full/nmeth.2972.html
Hope this helps!
Best wishes,
Michal
Hi Sylvia,
Probes described in this paper (recommended by Michal): http://www.nature.com/nmeth/journal/v11/n7/full/nmeth.2972.html are working with most of the mammalian cells. You can check SI for the details. Some groups have already tried on mouse oocytes and seem to be happy. However, it is important not to overdose the probe since it will lead to toxic effects.
Greetings,
Grazvydas
Hi Sylvia,
as Michal pointed out, the cell permeable probes described in that paper (I'm one of the authors) have a good chance to work in your system (especially if the ZP has been removed). Furthermore, the probes are commercially available through the company Spirochrome.
Best
Luc
Thank you Michal. That was very helpful information. Looks like there is some potential there and have contacted the company.
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