| Part ⅠThe exploration for quantitative detection of SEAPReporter gene assays system have become an important tool for the study of gene regulation.The expression of reporter gene can reflect the regulation of target gene. Reporter genes include luciferase, chloramphenicol acetyltransferase, fluorescent proteins, etc. However, tissues sampling or cell lysis are required to assay these reporter genes, which is inconvenient for continuous detection. The use of secreted alkaline phosphatase (SEAP) which can be secreted into the extracellular can solve this problem. Beside, the assay technology of SEAP is easy to be handled and low-cost. SEAP is one of the most widely used reporter genes。Generally, the assay of SEAP reporter gene is depended on the absorbance of SEAP which is detected by a microplate reader. However, the experimental conditions and other factors may affect the detection results. So it is difficult to comparate the gene expression under different experimental conditions, which has limited the application of SEAP. Therefore, a method for quantitatively detecting the expression of SEAP is immediately required. We have successfully established a method to calculate the quality of SEAP protein by detecting its absorbance, which can be used to better compare the analysis results of SEAP under different experimental conditions. Our studies provide better support for the application of SEAP in the future.Method:At first, we have established the standard curve of p-nitrophenol, which is the product of SEAP reaction. According to Lambert-Beer’s law:A=ε±c×d,We have calculated the molar extinction coefficient of p-nitrophenol under our experimental conditions. With the specific activity of SEAP, we found the formula between the absorbance value and SEAP quality. The formula has been successfully used for detecting the quality of SEAP in cell supernatants and mouse serum.Results:We have established a method to calculate the quality of SEAP protein by detecting its absorbance, which can be used under different experimental conditions. We have established the formula which can be used to calculate the Concentration of SEAP (μg·L-1):c=△A/min×0.033×dilution factor. Based on the formula, we can calculate the quality of SEAP(μg):m=△A/min×33.35×dilution factor×Sample volume. The formula has been successfully used to detect the quality of SEAP in supernatants and mouse serum. We validated the formula in vivo experiment of mice, and we found the expression of SEAP decreased with time after the hydrodynamic tail vein (HTV).Conclusion:We have established a method for quantitatively detecting the expression of SEAP, which improved the absorbance detection of SEAP. Our methods support the application of SEAP in the future. Part ⅡA preliminary exploration for long-term gene expression mediated by PB transposon and Tol2transposonTransposons are discrete DNA fragments that have the ability to move to other regions of the genome through a "cut-and-paste" mechanism, which process is called transposition. Transposon has become a research tool in genetics for applications in insertional mutagenesis and transgenesis. Viral vectors can be used to integrate their genetic cargo into the genome, and the gene can express in a long-term. However, viral vectors are expensive to prepare and its cargo size is small which limited the application of viral vectors. DNA can also be introduced into the genome by Transposon for a long-term expression. Moreover, the plasmid-based transposons have the advantage that it can be prepared with low cost and have a larger cargo size. We use Tol2transposon and piggyBac transposon which are broadly used to transfect the CHO and HEK293T cells in order to explore the expression of the gene carried by the transpon.Method:At first, we used PB transposon and Tol2transposon which can express fluorescent protein to transfect CHO cells. After the transfection, we observed the expression of fluorescent protein at different time by a fluorescence microscopy. Next, we transfected HEK293T and CHO cells with PB transposon and Tol2transposon, which can express SEAP reporter gene. The cell culture supernatant was collected at different time after the transfection for detecting the expression of SEAP. Though the stueies, we explored the long-term expression of the gene mediated by transposon.Results:After the CHO cells transfected by PB transposon and Tol2transposon which can express fluorescent protein, we found that the fluorescent protein continuously expression for a month. Similarly, we found that both the PB transposon and Tol2transposon can also mediate SEAP stable expression. In the studies, we also found that the transposition of PB transposon is more efficient than Tol2transposon. Conclusion:After transfected CHO cells and HEK293T cells by the transposon system, we found that transposon can mediated gene expression in a long-term, which provide that transposon system can be used for researching the long-term gene expression. |
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