A large-scale screening of Minos-inserted mutants and analysis of genes involved in the mutation in the chordate Clone intestinalis

玻璃海鞘提供了正向遗传学的希望，因为它们的生命周期相对较短，从受精到F1胚胎为8-12周，并且有可能在雌雄同体中诱导自花受精。紧凑的基因组还可以提供完成导致可见表型的突变的机会。在玻璃海鞘中，成功的生殖系转基因与Tc 1/水手超家族转座子，米诺斯，已经建立了我们自己。本研究的目的是分离尽可能多的Minos插入突变体，并确定负责突变的基因。本研究还旨在将各种转基因技术引入玻璃海鞘系统。在2005-2008年期间，我们已经分离了大约50个突变株系和20个转基因株系。突变株系包括游泳幼鱼（sj）1-4、气球（bal）和尾退化失败（tail retraction failed）。这些突变体均表现出变态过程异常。有趣的是，sj-I突变表型是由Minos插入到编码纤维素合酶的基因Ci-CesA的启动子区中而产生的。我们现在正在使用微阵列技术分析突变的分子机制。转基因系包括在胚胎肌肉、脊索或神经系统中具有GFP表达的那些。这些为进一步研究脊索动物基因的表达和功能提供了有用的标记系。

Ciona intestinalis offer the promise of forward-genetics since their life cycle is relatively short, 8-12 weeks from fertilization to Fl embryos, and it is possible to induce self-fertilization within hermaphrodites. The compact genome may also offer opportunity to accomplish mutations leading to visible phenotypes. In Ciona, successful germ-line transgenesis with the Tc1/mariner superfamily transposon, Minos, has been established by ourselves. The aim of the present study is to isolated Minos-inserted mutants as many as possible, and to identify genes that are responsible for the mutation. The present study is also intended to introduce various transgenic techniques into the Ciona system. During the period of 2005-2008, we have isolated approximately 50 mutation lines and 20 transgenic lines. The mutation lines include swimming juveniles (sj) 1-4, balloon (bal), and tail regression failed (trf). All these mutants showed abnormal process of metamorphosis. Interestingly, the sj- I mutant phenotype arises form the insertion of Minos into the promoter region of Ci-CesA, a gene encoding cellulose synthase. We are now analyzing molecular mechanisms involved in the mutations using microarray technique. Transgenic lines include those with GFP expression in embryonic muscle, notochord, or nervous system. These provide useful marker lines for further studies of the expression and function of chordate genes.