CHAPTER 7 - GENETIC METHODS
(Source: C. Walker)
Gamma rays are an effective mutagen for zebrafish embryos. Gamma-rays are known to produce many kinds of chromosomal damage such as small deletions, large deletions, chromosome inversions and rearrangements. We have used gamma-rays to produce mutations in three developmental stages of zebrafish: sperm, eggs, and 3 h blastulae. In all cases, the mutagenized embryos are raised to adulthood and the germ line is screened for interesting new mutations. The specific site mutation frequency is very similar for all three stages2,3.
Presently, we use gamma-rays to mutagenize at the sperm stage and at the blastula stage.
- Eggs 4 x 10-5/chromosome set/rad
- Sperm 4 x 10-5/chromosome set/rad
- Blastulae 1.2 x 10-5/chromosome set/rad
Sperm are pooled from a number of wild-type males into Hank's solution at the same concentration used for Embryo
Production By In Vitro Fertilization. Keeping
the sperm on ice, the sperm is put into the
chamber of a Gammatory-M irradiator
with a cesium-137
source and given a dose of approximately 250-300
rads. The irradiated sperm is used to fertilize
wild-type eggs. Approximately 25% of the embryos
adulthood. The advantages of gamma-irradiating
The sperm does not have as much non-chromosomal material to be damaged by the gamma-rays as eggs.
The mutation is present in 25%-50% of the germ line of the mutagenized female and, therefore, can be recognized and recovered easily.
Gamma-ray sperm do not tend to produce male adults as frequently as gamma-ray embryos do.
gamma-rayed 3 h blastulae
Wild-type eggs are squeezed and fertilized by wild-type sperm. The time of fertilization is noted. At 120 min, the embryos are irradiated in a small glass vial at a dose of 250-300 rads. Batches of eggs fertilized 4 min earlier and later can be pooled before irradiation. Survival ranges between 25%-50%.
A high percentage of males develop from gamma-irradiating blastulae. The percentage is linear with the dose (250-300 rads produces about 75-80% males).
Blastula stage embryos contain several germ line progenitor cells, from 1 to 104, providing more chromosome sets as targets for mutagenesis. The mutant clone size ranges from 5%-50% of the haploid germ cells.
We have found that embryos older than 3 h are considerably more resistant to gamma-rays as a mutagen suggesting that there may be a repair mechanism at work after cleavage stages.
It is presently unknown whether gamma-rays damage the sperm DNA in the same manner that they damage blastula DNA.
The Zebrafish Book