Notas de aplicación

Contemporary research on tardigrades

Dr. Kazuharu Arakawa at Keio University’s Laboratory for Advanced Biosciences et al. are conducting cutting-edge research to elucidate the ecology of tardigrades. How do tardigrades enter a state of anhydrobiosis (almost completely dehydrated dormancy) to withstand various extreme stresses when the environment is dry, and how can they be “brought back to life” by watering? Dr. Arakawa and colleagues are trying to answer these questions at the molecular level. Although genome sequencing and analysis of unique proteins have been carried out so far, deeper investigation has been very limited because there was no way to introduce foreign genes such as GFP into individual tardigrades or cells. Therefore, Dr. Arakawa et al. extracted the regions necessary for the expression of various genes from the tardigrade genome and developed tardigrade-specific gene expression vectors. When the vector, named TardiVec, was introduced into tardigrades using microinjection, expression of exogenous genes such as GFP was confirmed in multiple species of tardigrades. Furthermore, the GFP signal was retained for more than 10 days.

Overview

In this application note, we used the AX/AX R Confocal Microscope to perform live imaging of tardigrades in which mEGFP reporter was expressed with ubiquitin promoter using the TardiVec system. Two vectors in which mEGFP is inserted downstream of the ubiquitin-derived promoter of Ramazzottius varieornatus (Eutardigrada : Parachela ) and Milnesium inceptum (Tardigrada: Apochela) were utilized. These vectors were microinjected into Hypsibius exemplaris (Eutardigrada : Parachela ).

To perform Z-stack imaging, we controlled the movement of the tardigrades with a PF127 solution that forms a gel at room temperature, mixed polystyrene microparticles to prevent the tardigrades from being crushed, and covered them with a cover glass.

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