Tests of different mutagenic regimes in zebrafish favor mutagenizing premei-otic germ cells in adult males (Fig. 1). This is because, in a classical diploid screen, use of sperm derived from postmitotic stages of spermatogenesis results in a mosaic germline in F1 founder fish and increases the number of crosses required between F2 siblings to drive mutations to homozygosity. Mutagenizing G0 males involves the following procedures:
1. Pretest males (4-8 mo optimal) for high fertility by single pair matings. Place males directly in ENU solution.
2. Mutagenize in ENU solution for 1 h at room temperature in a darkened fume hood. The fish are stressed and tend to leap out of containers. A darkened quiet environment seems to minimize this behavior.
3. Transfer to a similar volume tank containing water from the fish facility at room temperature. Allow to recover for 6-8 h at this temperature.
4. Finally, transfer to a large 5-L tank in the fish facility.
5. Following a 1-wk recovery period, the treatment is repeated two further times, each followed by 1 wk of recovery. This recovery period may be shortened, but may lead to an increase in lethality. In a typical mutagenesis, only 30-50% of mutagenized fish survive all three treatments with increasing lethality per treatment.
6. Fish are then mated several times in a 4-6 wk period to remove mutant, mosaic postmeiotic sperm cells.
7. To test the efficacy of the mutagenesis, these G0 males (Fig. 1) are then crossed to a tester strain female, usually a strain carrying a homozygous viable pigment mutant, such as golden (gol). An effective mutagenesis should reveal a pigmentless embryo at a specific locus frequency of approx 1/500 (8,9).
8. These embryos may be raised as F1 founder fish or their G0 fathers crossed to wild-type females of a required strain to generate F1 founders.
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