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Exposure of insect cells to subzero temperatures typically leads to cell membrane disruption and lethal intracellular ice formation. This study seeks to examine the cryoprotective value of trangenically expressing a bacterial ice nucleation protein (INP) in Spodoptera frugiperda (Sf-21) cells. The bacterium Pseudomonas syringae naturally produces a membrane-bound INP (inaZ), capable of structuring water and initiating ice formation at temperatures as high as -2 °C. I hypothesized that intracellular expression of a genetically altered form of inaZ (PsINP) in Sf-21 cells will mediate highly regulated ice nucleation when cells are cooled to -80 °C in a slow, controlled manner. I also predicted that cells expressing PsINP (Sf-21-PsINP) will maintain cell membrane integrity in greater proportions than wildtype cells (Sf-21-WT). Following one freeze-thaw cycle, 60% of Sf-21-WT cell membranes remained intact, while 72% of Sf-21-PsINP cells maintained membrane integrity. This difference is statistically significant, and indicates that PsINP expression helps to prevent cryoinjury during freezing, and positively impacts cell viability following thawing.
Harder, Avril M., "Intracellular Expression of an Ice Nucleation Protein Reduces Cryoinjury in Insect Cells" (2012). Honors Theses. 1.