Functional Expression of Saccharomyces cerevisiae NADH-quinone Oxidoreductase (NDI1) Gene in the AML12 Mouse Liver Hepatocytes for the Applying Embryonic Stem Cell
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서지정보
ㆍ발행기관 : 한국동물번식학회
ㆍ수록지정보 : Reproductive & developmental biology / 35권 / 4호
ㆍ저자명 : Byoung Boo Seo, Humdai Park
ㆍ저자명 : Byoung Boo Seo, Humdai Park
목차
ABSTRACT INTRODUCTION MATERIALS AND METHODS Preparation of rAAV-NDI1 Cell Culture and rAAV-NDI1 Infection Viability Measurements Immunofluorescence Isolation of Mitochondria and Mitochondrial Membrane Fraction O2 Consumption Measurements Other Analytical Procedures Materials Statistical Analysis RESULTS Functional Expression of the NDI1 Gene in AML12 Mouse Liver Hepatocytes Effect of the Ndi1 Expression on the Electron Transfer Activity Western blotting Result of the Yeast NDI1 in AML12 Cells Effects of Complex I Inhibitors on Cell Growth DISCUSSION REFERENCES영어 초록
Mitochondria diseases have been reported to involve structural and functional defects of complex I-V. Especially, many of these diseases are known to be related to dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I). The dysfunction of mitochondria complex I is associated with neurodegenerative disorders, such as Parkinson's disease, Huntington's disease, and Leber’s hereditary optic neuropathy (LHON). Mammalian mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) is largest and consists of at least 46 different subunits. In contrast, the NDI1 gene of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. The Saccharomyces cerevisiae NDI1 gene using a recombinant adeno-associated virus vector (rAAV-NDI1) was successfully expressed in AML12 mouse liver hepatocytes and the NDI1-transduced cells were able to grow in media containing rotenone. In contrast, control cells that did not receive the NDI1 gene failed to survive. The expressed Ndi1 enzyme was recognized to be localized in mitochondria by confocal immunofluorescence microscopic analyses and immunoblotting. Using digitonin-permeabilized cells, it was shown that the NADH oxidase activity of the NDI1-transduced cells was not affected by rotenone which is inhibitor of complex I, but was inhibited by antimycin A. Furthermore, these results indicate that Ndi1 can be functionally expressed in the AML12 mouse liver hepatocytes. It is conceivable that the NDI1 gene is powerful tool for gene therapy of mitochondrial diseases caused by complex I deficiency. In the future, we will attempt to functionally express the NDI1 gene in mouse embryonic stem (mES) cell.참고 자료
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