Fig. 2.

Outline of mutagenesis in zebrafish used for TILLING and forward genetic screening. Both retroviruses and the mutagen ENU can be used to create germline mutations in zebrafish. For retroviral-based mutagenesis, a DNA construct containing the retrovirus is injected into newly fertilized zebrafish embryos. These injected embryos are grown to adulthood (2–3 months), resulting in G₀ adults that are mosaic for germline mutations. G₀ adults are then outcrossed to wildtype adults to generate G₁ adults that are heterozygous for different genetic lesions. In chemical mutagenesis, adult males are treated with a chemical mutagen such as ENU. These mutagenized males are crossed to wildtype adult females to generate G1 adults that are heterozygous for different genetic lesions. For a forward genetic screen (ENU or retroviral-based mutagens), G₁ adults are crossed to wildtype animals, providing a pool of G₂ adult carriers. G₂ adults are incrossed, and G₃ larvae are screened for a phenotype of interest. When mutagens are used to screen for a specific genetic lesion (in the case of TILLING), G₁ adults or sperm stored in a library from G₁ males are screened for mutations. After identifying a specific mutation, the identified G₁ adult is crossed to wildtype to generate G₂ adults harboring that mutation. Two G₂ adults with the lesion of interest are then incrossed and screened for phenotypes. Blue and green represent distinct genetic lesions in mutagenized fish