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Differences in Injection between X. tropicalis and X. laevis

Microinjection can be easily done in X. tropicalis although it does require some changes to laevis protocol to make it straightforward.

1. X. tropicalis lays eggs about 4 hours after a boost of hCG. So, there is at least a four hour delay between when an investigator gets to the lab and when he can inject. We have been unable to alter this significantly either shorter or much longer by manipulating temperature or the amount of hCG injected. Unlike X. laevis, X. tropicalis embryos cannot be injected "first thing in the morning".

2. In vitro fertilization is not as efficient in trops compared to laevis. We routinely use both testis for fertilization. Additionally we select males that have prominent nuptial pads. In our experience, males without nuptial pads fertilize very poorly. Fortunately, males can be raised in large numbers and housed in a much smaller space than laevis.

3. X. tropicalis embryos are much softer than X. laevis embryos. Initially, we felt this was due to their sensitivity to cysteine. However, cysteine does not appear to be problematic, and we do cysteine for up to 20 minutes without much problem. Because of the softness, we tested multiple ficoll concentrations. Two things seemed to help the most. First, we did an aggressive selection for females who produced the firmest eggs. Second, we try to keep the embryos in 3% ficoll as long as possible before injection.

4. X. tropicalis embryos whose jelly coats are removed by cysteine have a loose sticky vitelline membrane, and often stick to the bottom of the dish, and thus may have gastrulation problems. The gastrulation defect appears to be a mechanical effect of being stuck to the bottom of the dish. In order to combat "stickiness," dishes are coated with 1XMBS+0.2%BSA for manipulating eggs. Finally, we selected against females producing embryos having gastulation defects. We now have a sizable population of females making high quality injectable embryos.

5. X. tropicalis do not have a "summer slump." We find laevis produce low quality eggs during the summer months, however X. tropicalis do very well in the summer. They do, however often have a "winter slump", during which poor eggs and low fertilizations are the norm.