Detection of mutations in transgenic fish carrying a bacteriophage lambda cII transgene target

Abstract

To address the dual needs for improved methods to assess potential health risks associated with chemical exposure in aquatic environments and for new models for in vivo mutagenesis studies, we developed transgenic fish that carry multiple copies of a bacteriophage lambda vector that harbors the cII gene as a mutational target. We adapted a forward mutation assay, originally developed for lambda transgenic rodents, to recover cII mutants efficiently from fish genomic DNA by lambda in vitro packaging. After infecting and plating phage on a hfl- bacterial host, cII mutants were detected under selective conditions. We demonstrated that many fundamental features of mutation analyses based on lambda transgenic rodents are shared by transgenic fish. Spontaneous mutant frequencies, ranging from 4.3 x 10(-5) in liver, 2.9 x 10(-5) in whole fish, to 1.8 x 10(-5) in testes, were comparable to ranges in lambda transgenic rodents. Treatment with ethylnitrosourea resulted in concentration-dependent, tissue-specific, and time-dependent mutation inductions consistent with known mechanisms of action. Frequencies of mutants in liver increased insignificantly 5 days after ethylnitrosourea exposure, but increased 3.5-, 5.7- and 6. 7-fold above background at 15, 20, and 30 days, respectively. Mutants were induced 5-fold in testes at 5 days, attaining a peak 10-fold induction 15 days after treatment. Spontaneous and induced mutational spectra in the fish were also consistent with those of lambda transgenic rodent models. Our results demonstrate the feasibility of in vivo mutation analyses using transgenic fish and illustrate the potential value of fish as important comparative animal models.

Figure 1

Mutant frequencies (± standard error) in whole fish exposed 1 h to 0, 60, or 120 mg/liter ENU and held 15 days before analysis. cII mutants were induced 3- and 4-fold above the mutant frequency in untreated fish at each concentration.

Figure 2

Mutant frequencies (± standard error) in liver and testes from fish analyzed 5, 15, 20, or 30 days after 1-h exposure to 0 or 120 mg/liter ENU. Mutant frequencies in liver increased insignificantly over mean background (4.4 ± 0.7 × 10⁻⁵) at 5 days (5.1 ± 0.8 × 10⁻⁵), 4-fold at 15 days (15.6 ± 1.8 × 10⁻⁵), 6-fold at 20 days (25.1 ± 2.3 × 10⁻⁵), and 7-fold at 30 days (29.8 ± 2.0 × 10⁻⁵). Mutant frequencies in testes increased 5-fold over mean background (2.0 ± 0.6 × 10⁻⁵) at 5 days (10.3 ± 1.1 × 10⁻⁵), 10-fold at 15 days (19.6 ± 1.2 × 10⁻⁵), and 9-fold at 20 and 30 days (17.5 ± 1.6 × 10⁻⁵). –○–, ENU-exposed liver; –●–, ENU-exposed testes; - -○- - control liver; - -●- -, control testes.