Overexpression of human proislet amyloid polypeptide in CHO cells and characterization of its aggregation properties: a cell culture model of protein aggregation in mammalian cells | ||
| Pathobiology Reserach | ||
| Article 3, Volume 12, Issue 1, 2009, Pages 23-42 PDF (270.77 K) | ||
| Authors | ||
| مریم حسنلو; حمیدرضا کلهر* ; Majid Sadeghizadeh; Majid Sadeghizadeh | ||
| Department of Molecular Genetics, Tarbiat Modares University | ||
| Abstract | ||
| Objective: To study the mechanisms involved in amyloid formation processes, we made a mammalian cell culture model of amylin aggregation and characterized its properties. Materials and Methods: Amyloid fibrils were extracted from CHO cells and binding affinity to Thioflavin T and Congo red was investigated. Then the apple-green birefringence of extracted fibrils was detected under polarized light to confirm the presence of aggregated protein in the extracts. To better investigate amyloid formation in CHO cells, we decided to overexpress an amyloidogenic protein in these cells. To do so, we amplified ProIAPP gene and subcloned it in EGFP-N1 expression vector. Then, CHO cells were transfected with EGFP-N1-ProIAPP and EGFP-N1 as a control. The phenotypes of around 100 transfected cells were characterized for several days after the transfection, using fluorescence microscopy. Additionally, the presence of amyloid structure in these cells was detected by Congo red staining under exposure to polarized light. Cell viability assay was performed using Trypan blue staining. Results: Low level natural amyloid was detected in CHO cells. In addition, the ProIAPP-EGFP transfected cells exhibited aggregated phenotype in which the cells with round morphology had far more aggregates than oval ones. Conclusion: We found amyloid fibrils in CHO cells at low level. Overexpression of amylin protein in CHO cells caused aggregation phenotypes. These cells can be used as a model of cell culture for studying protein aggregation. Amyloidogenic properties of this protein could immensely help us to study the mechanisms that are involved in amyloid formation in mammalians including humans. | ||
| Keywords | ||
| Amylin; Aggregation; cell culture model | ||
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