Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan.
Dev Growth Differ. 2010 Feb;52(2):245-50. doi: 10.1111/j.1440-169X.2009.01168.x. Epub 2010 Jan 20.
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.
表型拯救实验在斑马鱼中被广泛应用，因为通过向胚胎注射 mRNA 可以很方便地研究突变表型的因果关系。然而，由于 mRNA 不稳定，这种策略仅对早期胚胎阶段的表型有效。对于后期发育阶段，使用 DNA 构建体来表达外源基因，但由于嵌合体问题通常无效。本研究试图通过 Tol2 介导的转基因来解决这个问题。作为一个模型案例，我们使用了 vlad tepes（vlt），一种斑马鱼 gata1 突变体，其表型在后期通过瞬时拯救实验从未得到过拯救。血细胞特异性的 gata1 转基因表达由其自身的启动子/增强子元件驱动。共注射含有 gata1 cDNA 和转座酶 mRNA 的 Tol2 供体质粒，可有效拯救 vlt 的无血细胞表型，即使在第 12 天的幼鱼中，当发生原始红细胞生成时，也会发生定型红细胞生成。因此，这种 Tol2 介导的拯救被认为是分析斑马鱼中突变表型的一种快速简便的方法。
