关键词: TALEN, 基因敲除, 文昌鱼, Hedgehog基因, 突变体

Abstract: The amphioxus is a promising animal model for evolutionary-developmental studies due to its key position on the animal phylogenetic tree. In the present study, we reported a genetically modified amphioxus strain on the Hedgehog (Hh) gene locus using the TALEN method. The result showed that our TALEN pair injection could bring about 34% mutations in the amphioxus Hh coding region. Further analysis on the F₀ gametic DNA revealed that the mutations had entered into gametes. So, we paired one F₀ male carrying an 8 bp deletion with a wild-type (WT) female, and carefully nursed the F₁ embryos up to adulthood. We then screened F₁ individually via analyzing their genomic DNA from a tiny tail tip, and obtained eight heterozygous mutants from the F₁ offspring. Moreover, our observation on the F₂ embryos generated by mating F₁ mutants also revealed that about 25% of early larvae developed aberrantly with head and tail curving ventrally, agenesis of the mesoblastic tissue under their anterior notochord, and no mouth opening. With the larva growth, deformities (such as twist of head and tail, mouth absent, ventrally localized endostyle and gill slits) became more severe, and eventually those malformed larvae died due to no food intake. Genetic analysis showed that all these deformed embryos were homozygous mutants and the ratio of Hh hetorozygotes vs WT agreed with Mondel's law. WT amphioxus larvae are asymmetric with the mouth on the left and gill slits on the right side. However, the homozygous mutant larvae became left-right symmetric with the gill slits on the ventral side, indicating a conserved role of Hedgehog signaling in establishing the left-right embryonic axis.

Key words: TALEN, gene knockout, amphioxus, hedgehog gene, mutant