Alternatively, those researchers that possess a large enough facility to generate and maintain large numbers of mutagenized families may opt for a classical diploid screen approach. In such a screen, mutations are homozygosed in the F3 generation. Advantages include a very low incidence of abnormal development per a given embryo in a brood, and mutations typically show Mendelian segregation. Recessive mutations will be a quarter of the progeny of the F2 intercross of the raised family (Fig. 1). Another drawback of haploid screens is that two generations are required before you recover the mutation as a diploid, because the squeezed female must be out-crossed and then the resulting progeny intercrossed before homozygous mutant embryos are produced. The F2 families raised in the diploid screen also provide a large number of fish immediately for analysis, since 50% of these fish should be heterozygous for the induced mutation (Fig. 1).
Diploid screening simply involves pair-mating fish of the F2 families, so that the statistical likelihood that two heterozygous fish for an induced mutation have been crossed is high. It is important to raise families of sufficient size and numbers of males and females to achieve this. Ideally 30-50 fish should be raised. Small families should be discarded as soon as it is realized that insufficient fish are present in the brood, since they will take up tank space and not provide sufficient fish to ensure detection of any mutations. Because 50% of fish should carry the mutation as heterozygotes in a given family, four to six crosses are usually sufficient to uncover mutations within a family. If the number of families is limited, more can be crossed to increase the certainty of detecting mutations. Pairs need to be kept separate until all embryo screening methods have been performed and, if no mutation is detected, can be returned to original tanks. Once it has been determined which mutations are to be kept, fish carrying those mutations must be identified from individual families by crossing to already identified fish. Noncarriers can be discarded, and tanks used for new F2 families.
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