Satoko Awazu

Germ-line transgenesis of the Tc1/mariner superfamily transposon Minos in Ciona intestinalis.

The tadpole larva of the basal chordate Ciona intestinalis has the most simplified, basic body-plan of chordates. Because it has a compact genome with a complete draft sequence, a large quantity of EST/cDNA information, and a short generation time, Ciona is a suitable model for future genetics. We establish here a transgenic technique in Ciona that uses the Tc1/mariner superfamily transposon Minos. Minos was integrated efficiently into the genome of germ cells and transmitted stably to subsequent generations. In addition, an enhancer-trap line was obtained. This is a demonstration of efficient, Minos-mediated transgenesis in marine invertebrates.

Culture of Ciona intestinalis in closed systems

Improvements in closed‐system culturing methods for marine invertebrates are important prerequisites for the generalized use of transgenic lines. We discuss here the effects of several closed‐system conditions on the growth and survival of the solitary ascidian, Ciona intestinalis. In Shimoda, close to the sea, a small‐tank system was used to ensure that tanks and systems were reasonably equipped, water exchange was rapid, and animals separated to minimize the risk of infection. In Gif‐sur‐Yvette, an inland site, we tried to determine the optimal conditions to limit handling operations, and to save artificial seawater by avoiding water pollution. A mixture of at least two types of live algae was better than any single‐organism diet. With these maintenance protocols, we were able to obtain several generations of Ciona intestinalis, including several transgenic lines. Because these systems make it easier to rear Ciona intestinalis in laboratories, they increase the potentialities of this model organism for research. Developmental Dynamics 236:1832–1840, 2007.

Application of Minos, one of the Tc1/mariner superfamily transposable elements, to ascidian embryos as a tool for insertional mutagenesis

As it has a simple genome structure, Ciona intestinalis is a good chordate species for studying the function of genes. To this end, it is a key requirement to introduce insertional mutagenesis using a transposable element to the ascidian system. The present study focuses on Minos, one of the Tc1/mariner superfamily transposons that is already used in a human cell line. By extrachromosomal excision and transposition assays, we found that Minos activity is very high in C. intestinalis. We also demonstrated the nuclear localization activity of Minos transposase in Ciona embryos. From these tests, we concluded that Minos transposase has complete activity when it is expressed in C. intestinalis, suggesting that Minos has the potential to be used for genome-wide insertional mutagenesis of C. intestinalis.

Transposon mediated transgenesis in a marine invertebrate chordate: Ciona intestinalis

Achievement of transposon mediated germline transgenesis in a basal chordate, Ciona intestinalis, is discussed. A Tc1/mariner superfamily transposon, Minos, has excision and transposition activities in Ciona. Minos enables the creation of stable transgenic lines, enhancer detection, and insertional mutagenesis.

A genomewide survey of developmentally relevant genes in Ciona intestinalis. IX: Genes for muscle structural proteins

Ascidians are simple chordates that are related to, and may resemble, vertebrate ancestors. Comparison of ascidian and vertebrate genomes is expected to provide insight into the molecular genetic basis of chordate/vertebrate evolution. We annotated muscle structural (contractile protein) genes in the completely determined genome sequence of the ascidian Ciona intestinalis, and examined gene expression patterns through extensive EST analysis. Ascidian muscle protein isoform families are generally of similar, or lesser, complexity in comparison with the corresponding vertebrate isoform families, and are based on gene duplication histories and alternative splicing mechanisms that are largely or entirely distinct from those responsible for generating the vertebrate isoforms. Although each of the three ascidian muscle types – larval tail muscle, adult body-wall muscle and heart – expresses a distinct profile of contractile protein isoforms, none of these isoforms are strictly orthologous to the smooth-muscle-specific, fast or slow skeletal muscle-specific, or heart-specific isoforms of vertebrates. Many isoform families showed larval-versus-adult differential expression and in several cases numerous very similar genes were expressed specifically in larval muscle. This may reflect different functional requirements of the locomotor larval muscle as opposed to the non-locomotor muscles of the sessile adult, and/or the biosynthetic demands of extremely rapid larval development.

Minos transposon causes germline transgenesis of the ascidian Ciona savignyi

An ascidian, Ciona savignyi, is regarded as a good experimental animal for genetics because of its small and compact genome for which a draft sequence is available, its short generation time and its interesting phylogenic position. ENU‐based mutagenesis has been carried out using this animal. However, insertional mutagenesis using transposable elements (transposons) has not yet been introduced. Recently, one of the Tc1/mariner superfamily transposons, Minos, was demonstrated to cause germline transgenesis in the related species Ciona intestinalis. In this report, we show that Minos has the ability to transpose from DNA to DNA in Ciona savignyi in transposition assays. Although the activity was slightly weaker than in Ciona intestinalis, Minos still caused germline transgenesis in Ciona savignyi. In addition, one insertion seemed to have caused an enhancer trapping. These results indicate that Minos provides a potential tool for transgenic techniques such as insertional mutagenesis in Ciona savignyi.

A genomewide survey of developmentally relevant genes in Ciona intestinalis. VIII. Genes for PI3K signaling and cell cycle.

Cell growth and cell divisions are two fundamental biological processes for cells in multi-cellular organisms. The molecules involved in these biological processes are highly conserved within eukaryotes, including plants and unicellular organisms such as yeast. However, some regulatory molecules seem to be innovated during animal evolution. Therefore, to understand how the ubiquitous systems have evolved or have been conserved, we examined genes for the phosphoinositide 3-kinase (PI3K) pathway that is important for cell growth, and genes for cell cycle regulation in the genome of Ciona intestinalis. It was found that the Ciona intestinalis genome contains all the essential constituents of the PI3K pathway. In addition, the class IB PI3K catalytic and regulatory subunits, which had not previously been known in animals other than mammals, were found in the Ciona genome. Similarly, all essential cyclins and CDKs were found in the Ciona genome, while cyclin G and cyclin L were likely to be independently lost in the ascidian lineage, which may be dispensable for the cell cycle. Cyclin F, which was previously known only in vertebrates, was not found in the Ciona genome. Therefore, this gene was probably innovated during the evolution of vertebrates to be involved in vertebrate-specific cell cycle regulation. Since Ciona is regarded as one of the most primitive extant chordates, the present analysis gives us an insight into how these fundamental biological genes are evolved or are conserved during chordate evolution.