We mutagenized the X. tropicalis genome with Gamma Ray irradiation of sperm and subsequent in vitro fertilization. Sperm mutagenesis has a few advantages. First sperm can be treated with a much higher dose of mutagen than eggs, embryos, or adults. Sperm have the least amount of non-chromosomal material to be damaged. Gamma rays create chromosomal deletions either small or large or chromosomal rearrangements such as translocations or inversions. In addition sperm mutagenesis is easy to do.

One disadvantage of sperm mutagenesis is that the animals produced are mosaic. Therefore phenotypes from the F1 animals may not show simple mendelian genetics. On the other hand, these mosaic F1 animals may be able to harbor more mutations without lethal dominant effects.

In our hands, gamma ray exposure results in a stereotypical gastrulation defect that is dose dependent. We have tried a number of different doses although the optimal dose remains uncertain.

Protocol for Gamma Mutagenesis of sperm:

Obtain two mature males with obvious nuptial pads

Prime and boost males and females with hCG

Remove both male testes and store on ice in 0.5 ml L15/CS(10%)

Get ready to mutagenize

1. Obtain a rack to hold the sperm in the Gammator. We used a 96 well PCR plate that we cut to a small enough size to fit in the Gammator.
2. Crush the testis and allocate the sperm solution into 0.2 ml PCR tubes that can stand nicely into the cut PCR plate without falling over. Save one for unirradiated control.
3. Expose in the Gammator for the desired dose. Our Gammator has a bar of Cesium-137 so the highest dose is in the center of the chamber. So, we tried to keep our sperm solution in the center.
4. Squeeze females and apply sperm. Monitor for fertilization
5. We recommend no cysteine or only a very light cysteine treatment just to separate the eggs. There will be a high mortality in this batch of embryos. It is considerably easier to simply place the embryos at low density and then just scoop out those embryos that hatch and are viable. Otherwise it would be hours of sorting dying embryos.