A chimeric HS4 insulator-scaffold attachment region enhances transgene expression in transfected Chinese hamster ovary cells

Abstract

Chinese hamster ovary (CHO) cells are one of the most commonly used expression systems for the production of recombinant proteins but low levels of transgene expression and transgene silencing are frequently encountered. Epigenetic regulatory elements such as the chicken β-globin locus control region hypersensitive site 4 (HS4) and scaffold/matrix attachment regions (S/MARs) have positive effects on transgene expression. In this study, a chimeric HS4-SAR was cloned upstream or downstream of an enhanced green fluorescent protein (eGFP) expression cassette in a eukaryotic vector, and the resulting vectors were transfected into CHO cells. eGFP was detected by flow cytometry. Real-time quantitative PCR (qPCR) was used to determine copy numbers of the stably transfected cells. And fluorescence in situ hybridization (FISH) was used to detect the status of vector in the host cell chromosome. The results showed that HS4-SAR positioned downstream of the expression cassette could enhance eGFP expression by 4.83-fold compared with the control vector. There may not be a relationship between transgene copy number and gene expression level. HS4-SAR did not appear to alter the integration of the transgene into the host cell chromosome or its position in the chromosome. We found a synthetic chimeric HS4-SAR positively increased transgene expression in CHO cells.

Keywords: Chinese hamster ovary; characteristic motif; enhanced green fluorescent protein; hypersensitive site 4–scaffold attachment region; scaffold/matrix attachment regions; transgene expression.

Figure 1

Synthesis of chimeric HS4‐SAR sequence and plasmid construction. According to reported chimeric HS4‐SAR sequence, HS4‐SAR was synthesized. The yellow (both light and dark) represents HS4 insulator, and the dark yellow is core sequence of HS4 insulator. The green represents interferon‐beta matrix association region. The red represents immunoglobulin matrix association region (A). The synthesis chimeric HS4‐SAR sequence was inserted into the upstream or downstream region of an enhanced green fluorescent protein (eGFP) expression cassette in pIRES‐eGFP to construct the pIRES‐sMAR5 and pIRES‐sMAR3, respectively (B). CMV, cytomegalovirus major immediate early; eGFP, enhanced green fluorescent protein; IRES, internal ribosome entry site; sMAR, synthetic matrix attachment region; SpA, simian virus 40 early polyadenylation signal.

Figure 2

Fluorescence microscopy of eGFP gene in transfected CHO cells after 48 h of transfection. (A) Micrograph of cells transfected with pIRES‐sMAR5 vectors (a), pIRES‐sMAR3 (b) vectors, and pIRES‐eGFP vectors (c). (B) Cell transfection efficiency was detected using eGFP antibody by flow cytometry. (C) Meanwhile, the transient expression levels of eGFP were obtained (*P < 0.05).

Figure 3

Analysis of the stable transgene eGFP expression. Cells were collected under G418 screening at day 20 post‐transfection. (A) Micrograph of cells that express different eGFP expression levels and transfected with pIRES‐sMAR3 vector (a) and pIRES‐eGFP vector (b). And the cell counts at different eGFP expression levels were compared (c). B) Flow cytometric analysis of transgene eGFP expression. After 20 days of transfecting vectors, recombinant protein expression stability was tested using flow cytometric analysis. The eGFP expression level was represented by the MFI. (C) The fold change of eGFP expression levels in cells transfected with pIRES‐sMAR3 and pIRES‐eGFP vectors was calculated (*P < 0.05).

Figure 4

Evaluation of stability of long‐term transgene expression. Stably transfected CHO cells were cultured in G418 (500 μg·mL⁻¹), and the cells were collected again and MFI by flow cytometry at days 90 after transfection. (A) Flow cytometric analysis of transgene eGFP expression levels. (B) The eGFP expression level was represented by the MFI. (C) Statistical analysis of recombinant protein expression rate (*P < 0.05).

Figure 5

Gene copy number was determined with fluorescent quantitative PCR. We collected the transfected cells that were cultured in G418 (500 μg·mL⁻¹) at 30 days post‐transfection. The copy numbers were tested using fluorescent quantitative PCR. And copy number's mean values differed between the vectors containing the HS4‐SAR and control (P < 0.05).

Figure 6

The status of plasmids in transfected CHO cells. At 30 days post‐transfection, the cells cultured in G418 (500 μg·mL⁻¹) were collected and tested by FISH analysis. pIRES‐sMAR3 (A), pIRES‐eGFP (B). 1: episomal; 2: integrated.