Gamma Ray Mutagenesis
We mutagenize the X. tropicalis genome with Gamma Ray irradiation of sperm followed by in vitro fertilization with non-mutagenized eggs. 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 small or large chromosomal deletions, 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:
Preparation for mutagenesis
1. Obtain a rack to hold the sperm in the Gammator. We used a 96-well PCR plate cut small enough to fit in the Gammator.
2. Crush the testis and allocate the sperm solution into 0.2 ml PCR tubes. The PCR tubes stand nicely into the cut PCR plate without falling over. Save one as un-irradiated 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.
5. We recommend no cysteine or 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 remove embryos that hatch and are viable. Otherwise hours of sorting dying embryos will be involved.