Abstract
In order to screen for developmentally active chromosomal domains during zebrafish embryogenesis, we generated transgenic fish by microinjecting two different lacZ reporter constructs into fertilized eggs. Transgenic fish were screened among the progeny of injected fish (Fo) crossed to non-injected fish. Groups of 15 to 20 progeny of each cross were tested for lacZ expression and/or transmission of injected sequences using PCR and Southern hybrizations. Progeny from 2 of 102 fish injected with super coiled constructs containing Rous sarcoma virus promoter sequences showed apparently spatially regulated β-galactosidase (β-Gal) activity. However, we were not able to detect this reporter construct in DNA from fins of F, fish. Injections of a linear reporter construct containing mouse heat-shock promoter sequences revealed transmission of injected sequences to Fi progeny in about 6% of cases (8 of 129 fish, tested with PCR). We found one ZacZ-expressing line that showed a spatially and temporally restricted expression of lacZ and, therefore, features typical characteristics of ‘enhancer trap’lines. In this line, lacZ expression starts at 16 hours post-fertilization in trigeminal ganglion cells. At about 24 hours lacZ expression can be detected in trigeminal ganglion neurons and Rohon-Beard neurons, indicating that the development of these two cell types shows common features. The reporter gene has integrated as a single copy. The founder fish was mosaic: 19% of its offspring (3 of 16 tested animals) carried the reporter construct in their fins; about 51% (13 of 27 tested animals) of the progeny of F, fish were β-Gal positive indicating full hemizygosity. We traced the heritability up to the 4th generation and showed that the reporter construct is stably integrated and inherited in a Mendelian manner. These results demonstrate that it is possible to generate “enhancer trap” lines in zebrafish, albeit with low efficiency.
