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De-Jellying Eggs

In order to protect the eggs and developing embryos, frogs produce a thick, sticky coating surrounding the egg called the jelly coat. This jelly coat must be removed before the embryo can be effectively manipulated or microinjected. Jelly coats are important for X. tropicalis development. We have noticed that completely de-jellied embryos often have difficulty gastrulating properly. We believe this is a mechanical problem. The vitelline envelope of X. tropicalis embryos is loose and sticky. Once stuck to the bottom of the dish, it appears to interfere with normal gastrulation.

However, simply coating the dishes with a thin layer of agarose prevents this problem. Therefore whenever we completely de-jelly the embryos, we raise them on agarose coated dishes until gastrulation is complete. Once gastrulation is completed, the embryos can be raised in regular petri dishes. If the embryos are not being microinjected, then a partial de-jelly can be very helpful. Often times due to the jelly coat, the embryos can be tightly stuck to each other. A partial de-jelly can separate the embryos and make it easy to transfer from dish to dish. The remaining jelly coat is enough to allow the embryos to gastrulate normally.

To de-jelly X. tropicalis embryos, immerse in 3% cysteine made up in 1/9xMR (pH to ~7.5-8.0 with NaOH) or water. Swirl the embryos gently and the jelly coat will slowly be removed. NOTE: Embryos being dejellied before the first cleavage may develop secondary axes if the embryos are swirled too vigorously. Therefore, if the dejellying is being done at the one cell stage, very gentle or no swirling is recommended. For embryos past the first cleavage, we swirl the embryos every so often in an erlenmeyer flask which greatly speeds the process.

For a partial de-jelly, immerse in cysteine just long enough until the embryos appear to be separate and sit next to each other in a monolayer. At this point the embryos should be easy to manipulate, and the jelly coat should still be visible around the embryo under the stereomicroscope. For a complete de-jelly, immerse in cysteine until the embryos pack tightly with each other without any clear space between the embryos. Again visualization under the stereomicroscope helps.

Initially we felt that X. tropicalis embryos were particularly sensitive to cysteine making the eggs soft and affecting gastrulation. Therefore we recommended very brief cysteine treatment. We no longer believe this to be true, and routinely treat in cysteine for as long as 20 minutes without problems. The softness and gastrulation defects appear to be due to other issues (see microinjection protocol for details).

Once the embryos are adequately de-jellied we rinse them multiple times with 1/9xMR and then they are ready for sorting.

Protocol from the Grainger site

This page contributed by Mustafa K. Khokha and Timothy C. Grammer