Poster #068, University of California Riverside
Engineering MDCK cells with Extremophile Protective Proteins
Mentors: Dr. Morgan, PI; Sara Nafar, Mentor; Daniel Bustamante
Extremophile organisms such as tardigrades and brine shrimp survive extreme conditions, including desiccation, radiation, and temperature extremes, by entering cryptobiosis, a reversible state of suspended metabolism. Fundamental to this survival ability are their unique stress proteins: MAHS protects mitochondrial function under stress; CAHS3, a flexible protein, forms a temporary gel-like network that provides physical support and slows cellular processes; and AfrLEA3m, a hydrophilic protein, prevents aggregation of sensitive mitochondrial enzymes during dehydration. To implement similar protective mechanisms in mammalian cells, we used a Sleeping Beauty transposon-based system to genetically engineer MDCK cells with MAHS, CAHS3, or AfrLEA3m, each tagged with an mScarlet fluorophore, alongside a control group. Rather than relying on viral delivery, this system uses SB transposase to excise the genes of interest from donor DNA constructs and integrate them directly into the host genome, enabling stable, long-term expression. The primary goal was to generate stable MDCK cell lines expressing CAHS3, MAHS, or AfrLEA3m, alongside a control line, as a foundation for subsequent stress-tolerance studies in mammalian cells.