Investigating the Role of Tardigrade Intrinsically Disordered Proteins in Desiccation Tolerance

Abstract

Fragile biological specimens, such as vaccines or human embryos in in vitro fertilization (IVF) clinics, are cryopreserved for storage. Cryopreservation is a tedious process with limited accessibility and can sometimes cause damage to specimens during freezing and thawing. Thus, room temperature storage of specimens through desiccation is an attractive alternative. However, this requires a method to safely desiccate and rehydrate samples. Tardigrades, microorganisms that are resistant to a variety of environmental stressors, express tardigrade disordered proteins (TDPs) such as cytosolic abundant heat soluble (CAHS) proteins during desiccation to facilitate survival. This project characterizes the use of CAHS proteins in mouse embryonic stem cells (mESCs) with the goal of eventually using these proteins to provide similar resistance to desiccation in mammalian cells. A CAHS protein under the Tet-On inducible promoter was integrated into the mESC genome using the piggy-Bac transposon system. The activation rates of this promoter were characterized by treating mESCs with varying levels of doxycycline. The impact of CAHS proteins on mESCs was also assayed using PIP-FUCCI, a fluorescent cell cycle reporter. Expression of the CAHS protein was dose-dependent, with higher concentrations of doxycycline causing more expression of the CAHS protein. Additionally, no significant changes to the cell cycle were detected in cells expressing the CAHS protein. This method of expressing CAHS proteins under an inducible promoter can be used in studies further analyzing CAHS-protein facilitated desiccation tolerance in mammalian cells and eventually for the room-temperature storage of medical specimens.