Efficient transient rescue of hematopoietic mutant phenotypes in zebrafish using Tol2-mediated transgenesis

Phenotypic rescue experiments have been commonly used in zebrafish since it is convenient to study the causality of mutant phenotypes just by injecting mRNA into embryos. However, this strategy is only effective for phenotypes at early embryonic stages due to mRNA instability. For later developmental stages, DNA constructs are used to express exogenous genes, while it is usually ineffective owing to the problem of mosaicism. This study attempted to solve the problem by using Tol2-mediated transgenesis. As a model case, we used vlad tepes (vlt), a zebrafish gata1 mutant, whose phenotypes have never been able to be rescued at later stages by transient rescue experiments. Blood cell-specific transgenic expression of gata1 was driven by its own promoter/enhancer elements. The co-injection of a Tol2-donor plasmid containing gata1 cDNA and transposase mRNA efficiently rescued the bloodless phenotypes of vlt even in day 12 larvae when definitive erythropoiesis took place with primitive erythropoiesis. This Tol2-mediated rescue is therefore considered to be a quick and easy method for analyzing the mutant phenotypes in zebrafish.