Date of Graduation
The technique of RNA interference was applied in two different systems. In system I, RNA interference was used to knockdown expression of Î±-aminoadipate Î´-semialdehyde synthase (AASS). AASS is the bifunctional enzyme containing the lysine Î±-ketoglutarate reductase (LKR) and saccharopine dehydrogenase activities responsible for the first two steps in the irreversible catabolism of lysine. A rare disease in humans, familial hyperlysinemia, can be caused by very low LKR activity and, as expected, reduces the lysine â€œrequirementâ€ of the individual. This concept was recreated in a murine hepatic cell line (ATCC, FL83B) utilizing RNA interference to achieve AASS mRNA knockdown. Cells were antibiotic selected for stable transfection of two plasmids that express different short hairpin RNA sequences for AASS knockdown. Compared to the wild type cell line, AASS mRNA abundance was reduced 79.0 Â± 6.4% (p<0.05), resulting in a 29.8 Â± 5.2% (p<0.05) reduction in AASS protein abundance, 46.6 Â± 7.7% (p<0.05) less LKR activity, and a reduction in lysine oxidation by 51.7 Â± 11.8%. To determine the effect of AASS knockdown on lysine requirement, cells were grown in media containing 12.5, 25.0, 50.0, 100, or 200 Î¼M lysine. Using a segmented model approach for growth rate analysis, the lysine requirement of the cell line with AASS silencing was 43.4 Â± 1.7 Î¼M, approximately 26% lower (p<0.05) than the lysine requirement of the wild type cell line. These results indicate AASS knockdown decreases the lysine requirement of the cell via a reduction of lysine catabolism through the saccharopine pathway, providing the initial proof in principle that RNA interference can be used to reduce the nutrient requirement of a system. In system II, RNA interference was used to knockdown urate oxidase (UOX) expression. Humans, birds, and higher primates do not express the uric acid degrading enzyme UOX and, as a result, have plasma uric acid concentrations higher than UOX expressing animals. Although high uric acid concentrations are suggested to increase the antioxidant defense system and provide a health advantage to animals without UOX, knockout mice lacking UOX develop pathological complications including gout and kidney failure. As an alternative to the knockout model, RNA interference was used to decrease UOX expression using stable transfection in a mouse hepatic cell line (ATCC, FL83B). Urate oxidase mRNA was reduced 66% compared to wild type, as measured by real time RT-PCR. To determine if UOX knockdown resulted in enhanced protection against oxidative stress, cells were challenged with hexavalent chromium (Cr(VI)) or 3-morpholinosydnonimine hydrochloride (SIN-1). Compared to wild type, cells with UOX knockdown exhibited a 37.2 Â± 3.5% reduction (p<0.05) in the electron spin resonance signal after being exposed to Cr(VI) and displayed less DNA fragmentation (P<0.05) following SIN-1 treatment. Cell viability decreased in wild type cells (P<0.05), but not cells with UOX knockdown, after treatment with SIN-1. These results are consistent with an increased intracellular uric acid concentration and an increased defense against oxidative stress.
Cleveland, Beth Marie, "The effect of Î±-aminoadipate Î´-semialdehyde synthase knockdown on the lysine requirement and urate oxidase knockdown on oxidative stress in a murine hepatic cell line." (2007). Graduate Theses, Dissertations, and Problem Reports. 8645.