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Xiaojun Yu, Wei Geng, Hongchuan Zhao, Guobin Wang, Yijun Zhao, Zhe Zhu, Xiaoping Geng
(Department of Hepato-Pancreato-Biliary Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China (mainland))
Med Sci Monit 2017; 23:5943-5950
Vectors are widely used to drive gene expression using a promoter. However, not all promoters are able to drive ectopic gene expression efficiently, including CMV promoter. Here, we report our data using CMV promoter for high-level gene expression in a B lymphoma cell line DG75.
MATERIAL AND METHODS: A plasmid (pcDNA3.1(+)) containing the CD21 gene driven under CMV promoter was constructed. The plasmid was stably transfected into a human B lymphoma cell line DG75 for cellular surface CD21 expression, and flow cytometry was used to monitor CD21 expression. CD21+ cells in the stable cell line were purified using anti-CD21 antibody-coupled Dynabeads for CD21-mediated antigen presentation experiment.
RESULTS: The percentage of CD21+ cells in newly generated stable DG75pcDNA3.1(+)CD21 cells was only 6.5% as determined by flow cytometry, which was unexpected and did not fit the requirements for further experiments. However, CD21+ cells could be purified to 100% using antiCD21 antibody-coupled beads. The percentage of CD21+ cells in purified cells can be kept at 95%, 82%, 42%, 15%, and 42% at 7 d, 14 d, 34 d, and 42 d after purification, respectively. Specific T cell response against CD21mediated antigen presentation can be activated successfully only when surface CD21 expression remains high.
CONCLUSIONS: A commonly down-regulated CMV promoter can be used to drive ectopic gene expression at a high-level in stable cell lines. Our results should facilitate future experimental design using other down-regulated promoters containing vectors such as SV40 and PGK1.