Generation of newly genetically modified lines in biomedical fish model species

Abstract

Zebrafish is a well-established animal model in neuroscience. The transparency of their eggs, high fertility, fast embryonic developmental, amenability to genetic manipulations, relative simplicity of the nervous system and high physiological and genetic homology to humans did not go unnoticed in the neuroscience field. The development of new transgenic tools and sophisticated imaging techniques, gave scientists the chance to generate genetically-modified zebrafish to visualize neurons, enabling the study, in vivo, of neuronal development, function and dynamics. The transgenic line Tg[nefma:KalTA4] uses KalTA4, a variant of Gal4 from the Gal4/UAS system. When crossed with a UAS reporter fish line, it labels nefma -expressing reticulospinal neurons, making this line a very convenient tool to study this neuronal population. However, the Gal4/UAS system faces a major challenge of reporter DNA silencing, due to UAS susceptibility to methylation. In this thesis, we used the QF/QUAS system and generated a new QF driver zebrafish line that labels nefma-expressing neurons, aiming to achieve a stable reporter expression across generations, with minimal toxicity. To this end, we used the CRISPR-Cas9 system to perform a knock-in of the QF sequence downstream of the nefma gene, via CRISPR-Cas9 nuclease-induced Homology -Directed Repair. This line was then crossed with a QUAS reporter fish line and the offspring screened for nefma-specific fluorescence expression. Even though no fluorescence was observed, we confirmed that the QF sequence was inserted into the fish genome and successfully transmitted to the offspring. Another fish species, Giant danio (Devario aequipinnatus) is a recent and promising model for neuroscience comparative studies with zebrafish. Comparative studies not only increase our knowledge of behavioral diversity, but also help distinguish between core aspects shared between species and specialized adaptations. Compared to zebrafish, Giant danio has a faster development, larger neurons and a rich behavioral repertoire. However, it lacks a toolkit similar to the existent for zebrafish. For example, both species are completely transparent during embryonic development, allowing the visualization and interference with neuronal activity. However, melanophores appear in the early stages of development, hampering the visual access to the brain. In zebrafish, this has been overcome by the creation of pigmentless mutant lines, but an equivalent model in Giant danio is missing. As such, a second objective of this thesis was to establish a new Giant danio model, a mutant line without melanophores. For this purpose, we used the CRISPR-Cas9 system to knock out the mitfa gene, which is involved in the development of melanophores. We designed and synthetized a sgRNA targeting a gene region that is conserved across different species. We tested it in vitro in Giant danio and in vivo in zebrafish, which has a higher breeding performance. No pigment loss was observed, and genotyping confirmed that neither of the zebrafish mitfa alleles were successfully mutated. The in vivo test has yet to be performed in Giant danio. Generation of a mitfa mutant line in Giant danio would significantly ease the imaging studies in this species.