Inducible System For Dominant Mutant Screening In Zebrafish

Forward genetic screen in model organisms is one of the major strategies for analyzing complex biological processes. In conventional genetic screens, chemicals or ionizing radiation or insertions of tagged-DNA are applied to create random mutations in genomes. This often results in full or partial loss-of-function mutations but may also occasionally create gain-of-function mutations. Although conventional genetic approaches have proven to be extremely powerful, these approaches do have their limitations. For examples, it takes three generations to get homozygous mutants, which is time-consuming and requires a lot of facilities; also numerous studies have shown that ectopic expression can also be informative towards the understanding of gene function; one key disadvantage is that conventional approaches prevent us from studying the gene function in the adult organism (such as its role in organ regeneration) in the cases where the gene under study is essential for early development and the mutation is lethal. Therefore, an inducible system that allows the mutant to grow to adulthood normally, and only then to alter the expression (down-or up-regulation) of the endogenous gene for gene function studies, is much required.Zebrafish (Danio rerio) represents an ideal model system, not only for studies of early development, but also, as zebrafish can regenerate most of their organ/tissues1,21,2, for organ regeneration studies. Successful application of chemical (N-ethyl-N-nitrosourea ENU) and insertional (retroviral and transposon) mutagens has established the zebrafish as a forward genetic model system.In this thesis, I develop an inducible system for dominant mutant screening based on Tol2-mediated insertional mutagenesis. This allows the screening for both embryonic developmental mutants and adult organ/tissue regeneration mutants. The system relies on Tet-on promoter driven sense or anti-sense expression of the tagged genes upon doxycycline-treatment, thus mimicking conditional gene overexpression or knockdown in heterozygous genetic background. We generated 300 independent transgenic lines for screening and identified 27 phenotypic mutants including 20 lines displaying embryonic developmental defects only,3 lines showing adult fin regeneration defects only, and 4 lines exhibiting both defects. A total of 58 disrupted genes, including 8 non-coding genes in the mutant lines, were identified to be either knocked down or overexpressed by their corresponding insertions. Among the 58 genes, fafl, grbl4, pcytlaa, nidogen2, sgcd and ap2bl have been previously found to play an important role in embryonic development. The phenotypes in these mutants mimic those in previous publications. The study demonstrates that the new system is a powerful tool for forward genetic screening, especially in adult vertebrates.