Figure 1

Figure 1

Genetic suppressor screens in zebrafish reveal additional mutations capable of rescue. (a) The bacterial artificial chromosome (BAC) transgenic construct containing a wild-type Tif1gamma locus and green fluorescent protein (GFP) driven by an actin promoter (Pactin) used in our recent genetic suppressor screen [2]. The transgene was injected into one-cell-stage embryos (right) to rescue the lethality of Tif1gamma mutant (mon) fish. (b) Schematic diagram of the suppressor screen. Stable transgenic fish are homozygous mutants for the endogenous tif1gamma locus (mon/mon) but retain viability because they are heterozygous for the transgene. The GFP marker on the transgene makes them green fluorescent. F0 males were mutagenized with ethylnitrosourea (ENU). In the F₁ generation, 25% of progeny were transgene homozygotes (Tg homo, mon/mon; Tg/Tg, bright green), 50% were transgene heterozygotes (Tg het, mon/mon; Tg/+, light green, in red circle), and 25% lacked the transgene (No Tg, mon/mon, gray). Only the progeny that were heterozygous for the transgene were raised to adults. The F₁ females were then squeezed to provide unfertilized eggs that were activated by UV-treated sperm. The UV treatment destroys the paternal DNA while still allowing fertilization. The resulting F₂ embryos were haploid and were subjected to in situ hybridization (ISH) at 22 hours post-fertilization for GFP and beta e3 globin probes. Transgenic embryos (mon;Tg) were positive for both probes, whereas non-transgenic embryos (mon) were negative for both probes. However, embryos that were negative for GFP but positive for globin indicated the presence of a genomic suppressor (sup) mutation. Modified, with permission, from [2].