Group led by the Exploratory Research Center on Life and Living Systems develop gene expression vector derived from tardigrade genome that realizes GFP‐fluorescence

A research group led by Project Assistant Professor Sae Tanaka of the Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, and Professor Kazuharu Arakawa of the Institute for Advanced Biosciences, Keio University, in collaboration with Professor Kazuhiro Aoki of the National Institute for Basic Biology, has developed 'TardiVec,' a DNA vector using genome‐derived sequences of the tardigrade, an animal that can withstand extreme environments, and succeeded in expressing green fluorescent protein (GFP) in the animal. By using the same vector, the researchers were able to confirm tardigrade‐specific gene expression and protein behavior. A video of tardigrades expressing GFP is available on YouTube (https://youtu.be/WylLl98I7Es). The results are expected to lead to shedding light on the molecular mechanism of the ability of tardigrades to enter the ametabolic state of anhydrobiosis (loss of almost all water in the body). The group's research was published in the January 24 issue of PNAS, Proceedings of the National Academy of Sciences.

Tardigrades are microscopic aquatic animals measuring 0.1‐1 mm in length that can live almost anywhere, including extreme environments such as Antarctica and the deep sea. More than 1,400 species have been reported, some of which are tolerant to desiccation through anhydrobiosis, where the moisture content in the body drops to below 3% when the surrounding environment dries out, resulting in an anabolic state. They have a lifespan of about two months in normal active conditions, but have been found to survive for 30‐40 years in anhydrobiosis, and can withstand environments such as high temperatures, high pressure and high doses of radiation, and will revive if they are given water.

Understanding the mechanism of anhydrobiosis is expected to lead to perfect dry storage, and has been the subject of much research, including genome decoding. However, there has been no way to introduce foreign genes into individual tardigrades and analysis has been limited.

To date, the research group has worked on deciphering the tardigrade genome, establishing breeding methods and analyzing gene functions using human cultured cells.

They have succeeded in developing a tardigrade‐specific gene expression vector by extracting the regions essential for the expression of various genes from the tardigrade genome. They have named it 'TardiVec (Tardigrade vector).'

When an aqueous solution incorporating GFP in the same vector was injected into tardigrades, the expression vector functioned in the tardigrade cells and the researchers confirmed fluorescence with GFP in live tardigrades. 'TardiVec' also maintains the tissue of expression of each gene of origin, and this characteristic revealed for the first time that the unique gene has a tissue‐specific gene expression pattern.

While the tardigrade genome has a similar number of genes to that of its closely related model animal, the centipede, about 40% of its genes are known to be unique protein genes. The researchers therefore thought it to be involved in anhydrobiosis as they had previously identified CAHS, MHAS, SAHS and Dsup as tardigrade endemic protein genes.

With this in mind, they examined tardigrades using 'TardiVec' the gene expression of CAHS and SAHS, which are thought to be associated with anhydrobiosis.

They found that SAHS genes are predominantly expressed in storage cells floating in the tardigrade body cavity, after which SAHS proteins are secreted extracellularly and spread throughout the body. In contrast, CAHS genes are predominantly expressed in body surface cells and CAHS proteins are found to localize to the cytoplasm of the cells in which they are expressed.

The fluorescence of GFP transduced with the vector can be observed from 48 hours onwards for approximately 30 days, confirming that fluorescence is maintained after recovery from anhydrobiosis.

"Anhydrobiosis is a very dynamic phenomenon," commented Tanaka. "Now that we can do live imaging, we expect to be able to properly reveal what phenomena are happening with our own eyes."

Journal Information
Publication: Proceedings of the National Academy of Sciences of the United States of America (PNAS)
Title: In vivo expression vector derived from anhydrobiotic tardigrade genome enables live imaging in Eutardigrada
DOI: 10.1073/pnas.2216739120