Date of Award


Degree Type


Degree Name

Master of Science




Dr. Richard Rozmahel

Second Advisor

Dr. Gary Shaw

Third Advisor

Dr. Jim Koropatnick


The SI00 calcium-binding protein A8 (S100A8) has been identified as a potential genetic modifier of the lung disease of cystic fibrosis (CF) patients as well as mice. S100A8 and its heterodimeric partner, S100A9, are expressed by neutrophils and monocytes and secreted to the extracellular milieu during inflammation where they act as potent chemokines for neutrophil recruitment. The aim of this study was to assess the efficacy of short-hairpin RNAs (shRNAs) in suppressing murine S100A8 expression in vitro as a preliminary model to the study of S100A8 knockdown in CF mice. Stimulation of murine macrophage-like P388D1 and granulocytic 32D cells failed to induce S100A8 expression. COS-7 (African green monkey kidney) cells, stably transfected with murine S100A8, showed S100A8 mRNA but no protein. Introduction of murine S100A9 into wild-type and COS-7 cells stable for S100A8 expression revealed S100A9, but no S100A8, protein. The fact that S100A8 protein was not detected contradicts, at least, the idea that S100A9 protein requires S100A8 for stability, although the opposite may also be true. Three different miR-30-based shRNAs targeting S100A8 and a non-specific shRNA (control) were evaluated for their knockdown efficacy. Two of the three hairpins tested (shRNA_mA8_65 and shRNA_mA8_250) were able to significantly reduce S100A8 mRNA levels compared the untreated and control-treated cells (p=0.001 and j9=0.033, respectively), achieving 62% and 25% suppression, respectively. This study has identified shRNAs that can specifically silence the expression (and achieve stable knockdown) of S100A8 in COS-7 cells over-expressing the S100A8 gene. This will be valuable for both in vitro and in vivo functional studies of S100A8; particularly, to study the effects of S100A8 suppression on the inflammatory lung phenotype in CF mice.



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