POAG disease-causing gene MYOC mutant vector and in the COS7 cells.doc

 PAGE \* MERGEFORMAT 13 POAG disease-causing gene MYOC mutant vector and in the COS7 cells [Abstract] Objective: To construct Pro370Leu mutation type Myocilin (MYOC) gene eukaryotic expression plasmid, and expressed in COS  7 cells to examine the characteristics of mutant protein expression. Methods: pGEM  T  MYOC in MYOC plasmid as template, using PCR -mediated site-directed mutagenesis techniques, get Pro370Leu mutant MYOC gene (mutant MYOC, mMYOC), and subcloned into the eukaryotic expression vector pDsRed2  N1, commanding pDsRed2  N1  mMYOC recombinant expression plasmid, and then restriction endonuclease enzyme digestion and DNA sequencing, and finally embedded liposome transfection method transfected COS  7 cells, fluorescence microscopy. using Western Blot analysis of protein secretion and detection of mutant protein on cell apoptosis. Results: After enzyme digestion and DNA sequencing, the first 370 codon CCG into CTG, fluorescence microscope observations indicate that mMYOC protein is only detected in the cytoplasm, the mMYOC by Western Blot analysis showed that the protein only in cell lysates by flow cytometry mutant transfected group a higher percentage of apoptotic cells was 6.63%. Conclusion: Pro370Leu mutant MYOC gene eukaryotic expression plasmid was successfully constructed, Pro370Leu mutant MYOC gene protein was secreted disability status, and has the tendency to promote apoptosis. [Keywords:] MYOC gene site-directed mutagenesis of apoptosis open-angle glaucoma 0 Introduction Primary open-angle glaucoma (primary open angle glaucoma, POAG) is one of the major blinding eye diseases, studies have found that POAG has a significant genetic disease. In 1997, the first time, Stone and other cloned genes that cause POAG in: Myocilin (MYOC) gene, the gene is by far the most studied and POAG-related genes have been found in dozens of disease-causing mutations associated with POAG, such as found in China Pro370Leu, T455KmMYOC genes, and