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Differences in injection of X. laevis and X. tropicalis

Microinjection can be easily done in X. tropicalis although it does require some differences in 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. X. tropicalis embryos cannot be injected "first thing in the morning" unlike X. laevis.

2. In vitro fertilization is not nearly as efficient in trops compared to laevis. We routinely use both testis for fertilization. Additionally we select males that have prominent nuptial pads. Our experience has been that males without nuptial pads fertilize very poorly. However, intensity of the nuptial pad color does not necessarily correlate with the percent fertilization. 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 that 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. Also embryos whose jelly coats have been completely removed often stick to the bottom of the dish and 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 L15/10% calf serum for manipulating eggs. And then once the jelly coats are removed, the embryos are kept on agarose coated dishes. Finally we selected against females that produce embryos that have gastulation defects. We now have a sizable population of females that make high quality injectable embryos.

5. X. tropicalis do not have a "summer slump." In our experience, laevis produce low quality eggs during the summer months. X. tropicalis does very well in the summer. There have been reports that trops do not produce good eggs in the winter, however. Our experience has been limited, and we'll re-evaluate this winter with our females that have been selected for high-quality egg production.