Title: Construction of Expression Vector pcDNA3.1/myc-His-DJ-1~(M26I) and Study of its Impaction on Cell Proliferation and Apoptosis in NIH3T3 Cells
Abstract: Objective To construct the recombinant expression vectors of pcDNA3.1/myc-His-DJ-1 and pcDNA3.1/myc-His-DJ-1M26I,and provid a basis for further study on the relationship of DJ-1M26I mutation and cell proliferation and apoptosis and establish transgenic animal model.Methods Using site-directed mutagenesis kit to make the 26th amino acid mutated,constructed pcDNA3.1/myc-His-DJ-1 and pcDNA3.1/myc-His-DJ-1M26I recombinant expression vectors.And then transfected them into NIH3T3 cells respectively using lipofectamine.Cells were selected with G418 at the level of 500μg/ml.Stable clones were identified on the DNA,RNA and protein levels.Using MTT assay and AnnexinV-FITC kit to detect the stable cloned cells' viability and apoptosis level.Results NIH3T3 cells transfected with recombinant plasmid pcDNA3.1/myc-His-DJ-1 or pcDNA3.1/myc-His-DJ-1M26I screened by G418.We obtained 1 and 3 positive clones of pcDNA3.1/myc-His-DJ-1 and pcDNA3.1/myc-His-DJ-1M26I transfected cells by PCR detected.The results of RT-PCR and western blot also showed the expression of DJ-1-His in pcDNA3.1/myc-His-DJ-1 and pcDNA3.1/myc-His-DJ-1M26I transfected cells.MTT assay demonstrated the NIH3T3 positive cells transfected with DJ-1M26I had the lower proliferation rate than normal NIH3T3 cells(p0.05).NIH3T3 positive cells carrying the DJ-1 genes showed no significant difference compared with the normal cells NIH3T3 cells.Apoptosis test indicated that the apoptosis rate of DJ-1M26I transfected cells was higher than normal NIH3T3 cells,however the apoptosis rate of the DJ-1 transfected cells was lower than normal NIH3T3 cells(p0.05).Conclusions Recombinant expression vectors pcDNA3.1/myc-His-DJ-1 and pcDNA3.1/myc-His-DJ-1M26I have constructed successfully.The NIH3T3 cells with stable expression of DJ-1 and DJ-1M26I have been constructed successfully.DJ-1M26I mutation can result in the apoptosis of NIH3T3 cells easily.
Publication Year: 2012
Publication Date: 2012-01-01
Language: en
Type: article
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