AI Article Synopsis
- The study aims to investigate how different promoters and matrix attachment regions (MAR) affect transgene expression in CHO cells.
- It combines various promoters with a MAR in expression vectors, transfects them into cells, and measures the expression of enhanced green fluorescent protein (eGFP) using flow cytometry and qPCR.
- Results reveal that the tested promoter is more effective than another, and the presence of MAR enhances transgene expression by increasing gene copy numbers.
Article Abstract
Objective: To analyze the effects of different promoters and matrix attachment region (MAR) on the expression of transgene in Chinese hamster ovary (CHO) cells.
Methods: The expression vector was constructed by the combination of beta globin MAR with the human cytomegalovirus immediate-early promoter and simian virus 40 promoter. These vectors were transfected into CHO cells,after 48 h,the transient expression of enhanced green fluorescent protein (eGFP) was observed; G418 was used to screen stably transformed cell lines,and the expression level of in CHO cells was analyzed by flow cytometry. The relative copy numbers of were analyzed by qPCR.
Results: Without expression vector,the expression of which was driven by promoter was stronger than that of promoter; could increase the expression level of driven by promoter,but did not show any enhancement in promoter. The expression level of which containing on both sides was stronger than that of on one side driven by promoter; After G418 screening,the expression level of containing driven by promoter wasunstable,the fluorescence gradually weakened,therefore,we only analyzed the expression vector stably expressing the gene driven by promoter by flow cytometry and qPCR. Compared with the expression vector without containing promoter,flow cytometry showed that the expression levels of on one and both sides with were increased by 9.85-fold and 12.94-fold,respectivley; The result of qPCR showed that the copy number of the gene without was set to 1,the copy number of the gene in the expression vector driven by with on one side and both sides were 3.68-fold and 9.25-fold,respectively.
Conclusion: The activity of promoter is stronger than that of promoter. can enhance the expression levels of transgene,which may be related to the increase of gene copy number.
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