Two human MARs effectively increase transgene expression in transfected CHO cells

Abstract

Matrix attachment regions (MARs) can enhance the expression level of transgene in Chinese hamster ovaries (CHO) cell expression system. However, improvements in function and analyses of the mechanism remains unclear. In this study, we screened two new and more functional MAR elements from the human genome DNA. The human MAR-3 and MAR-7 element were cloned and inserted downstream of the polyA site in a eukaryotic vector. The constructs were transfected into CHO cells, and screened under G418 to produce the stably transfected cell pools. The expression levels and stability of enhanced green fluorescent protein (eGFP) were detected by flow cytometry. The transgene copy number and transgene expression at mRNA level were detected by quantitative real-time PCR. The results showed that the expression level of eGFP of cells transfected with MAR-containing vectors were all higher than those of the vectors without MARs under transient and stably transfection. The enhancing effect of MAR-7 was higher than that of MAR-3. Additionally, we found that MAR significantly increased eGFP copy numbers and eGFP gene mRNA expression level as compared with the vector without. In conclusion, MAR-3 and MAR-7 gene can promote the expression of transgene in transfected CHO cells, and its effect may be related to the increase of the number of copies.

Keywords: Chinese hamster ovaries cell; gene expression; matrix attachment region; transgene silencing.

Figure 1

Analysis of eGFP MFI in stably transfected CHO cell line. (A) The eGFP MFI was measured after culturing cells 20 days under G418 pressure. Each value represents the average and standard deviation of three independent stably transfected pools. (B) Fold statistical analysis results of expression level, and the eGFP MFI were normalized to a control vector without MAR

Figure 2

Analysis of the relative eGFP mRNA levels and eGFP copy number in stably transfected cell pools. Fluorescent quantitative PCR was used to measure relative eGFP mRNA levels and gene copy numbers. The 2^−∆∆Ct method was used to calculate relative values. The mRNA levels and eGFP gene copy numbers were normalized to the control whose value was set to 1. Three independent experiments were performed in this study. Standard error of the mean (SEM) is indicated (*P < 0.05)