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This page is meant to help beginners -typically undergraduate or graduate student and post-docs- willing to ascidian development using molecular tools to address questions about gene activities and/or tissue-specific developmental processes.
obtaining and keeping healthy gravid adults
Most research with ascidian embryos, the species Ciona intestinalis being taken as an example here, are performed on samples obtained by in vitro fertilization of gametes surgically extracted from wild adult animals.
Setting-up a sea water tank
The details here can be arranged with a tank provider, the basic principles are that the tank needs to be equipped with appropriate pump, biological filter, skimmer, cooler and CONSTANT ILLUMINATION, this is critical to avoid spontaneous spawning at dawn. In addition, the animals are preferably place in hanging baskets for easy access.
The biological parameters of the tank will take a few weeks to adjust and must be monitored. Commercially available kits for pH, KH, nitrates, nitrites can help determine when an acceptable equilibrium has been attained.
Gravid adults can be kept for two to three weeks, care must be taken to remove dead bodies, which would quickly pollute the tank and affect their healthy roommates. (dispose of dead animals as per your university or institute's standards).
The first task is te determine the species to work with. For labs working with Ciona intestinalis, care should be taken to determine whether the most readily available type is A or B. Type A is thought to prevail in the Pacifique ocean and has provided the genomic DNA for whole genome sequencing. Type B is thought to inhabit mostly the northern european coasts.
Two options allow researchers to obtained gravid Ciona intestinalis adults: either field-collection or purchasing to specialized vendors.
For field-collection, permission must first be obtained by the department of environmental conservation, then a trip to a local harbour can be organized: just bring a cooler, buckets, and not-too-precious clothes! Animals can then be found by searching underneath floating devices, in the shade. The chance of success depends on the season: for example, in the Pillar point harbour, Half Moon Bay (California), Ciona adults flourish from may to november, but can almost not be found from january to march. An alternative to random searches is to immerse trays attached to water-resistant ropes and let animals populate them for a few weeks. These will be covered with a wide variety of marine invertebrates, but Ciona settlement can be facilitated by removing non desired species regularly.
fertilization, dechorionation and embryo culture
database search, sequence analysis and preparation of molecular tools
electroporation and microinjection of Ciona eggs
fixing, staining and mounting embryos