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Abstract #13636 Published in IGR 8-2
Comparative study of eukaryotic cell expression of wild-type and Pro370Leu mutation type myocilin gene
Wang J-W; Sun H-M; Ji J; Li X-R; Zheng J-QZhonghua Yi Xue Za Zhi 2006; 86: 700-704
OBJECTIVE: To compare the eukaryotic cell expression of wild-type myocilin (MYOC) (wMYOC) gene and Pro370Leu mutation type MYOC (mMYOC) gene so as to understand the mechanism of primary open angle glaucoma (POAG). METHODS: HeLa cells were cultured and then transfected with the vector pEGFP-N3-wMYOC, recombinant plasmid with wild type MYOC gene and enhanced green fluorescein gene, or the vector pDsRed2-N1-mMYOC, recombinant plasmid with mutation MYOC gene and red fluorescein gene, respectively, or co-transfected with these 2 plasmids. Corresponding blank vectors pEGFP-N3 and pDsRed2-N1 were used as markers. Fluorescence microscopy was used to observe the localization of red fluorescence and green fluorescence. Laser co-focusing microscopy was used to observe the effect of co-transfection. The green and red fluorescein antibodies were examined by Western blotting. RESULTS: Green fluorescence was observed in the cytoplasm of the HeLa cells transfected with the blank vector pEGFP-N3, in an even distribution; and red fluorescence was observed in the cytoplasm of the HeLa cells transfected with the blank vector pDsRed2-N1, in an even distribution too. The cells transfected with pEGFP-N3-wMYOC showed evenly-distributed green fluorescence in the cytoplasm, and the cells transfected with pDsRed2-N1-mMYOC showed red fluorescence in the cytoplasm in a state of aggregation. Both red and green fluorescence could be seen in the cells co-transfected with pEGFP-N3-wMYOC and pDsRed2-N1-mMYOC, both in a state of aggregation and co-localization. Laser co-focusing microscopy showed the same results. Protein with the relative molecular weight of 83 000, identical to that of the recombinant protein of wMYOC protein and green fluorescein, could be found in the culture fluid and cell lysate of the pEGFP-N3-wMYOC-transfected cells; however, could be found in the lysate only but not in the culture fluid of the co-transfected cells. Protein with the relative molecular weight of 81 000, identical to that of the recombinant protein of mMYOC protein and red fluorescein, could be found in the cell lysates of the pDsRed2-N1-mMYOC -transfected cells and the pEGFP-N3-wMYOC and pDsRed2-N1-mMYOC co-transfected cells, but not in the culture fluid of both cells. CONCLUSION: Both the WMYOC and mMYOC genes can be expressed and localized in the cytoplasm. mMYOC protein shoes a tendency of aggregation and disorder in secretion, and affects the expression and secretion of wMYOC, thus influencing the factions of the trabecular meshwork and causing POAG. LA: Chinese
Dr. H.-M. Sun, Ophthalmology Center, Tianjin Medical University, Tianjin 300070, China
Classification:
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
