Human rhinovirus internal ribosome entry site element enhances transgene expression in transfected CHO-S cells

Figures

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  Figure 1. Schematic diagram of vectors pIRES-EGFP (A) and pIRES-EGFP1-10 (B) containing different size of IRES fragment. CMV, human cytomegalo virus immediate early gene promoter; EGFP, enhanced green fluorescent protein; IVS, synthetic intron; IRES, internal ribosome entry site.

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  Figure 2. Fluorescence profile of EGFP in CHO-S cells. CHO-S cells transfected with vectors containing IRES elements from different resources were measured using FACS after 48 h transfection. (A) Representative of high EGFP fluorescence profile in cells with the pIRES-EGFP-9. (B) Representative of medium EGFP fluorescence profile in cells with the pIRES-EGFP-4. (C) Representative of low EGFP fluorescence profile in cells with the pIRES-EGFP-2. (D) Representative of untransfected cells. Scale bar, 100 μm.

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  Figure 3. The EGFP protein levels in CHO-S cells determined by flow cytometry. Representative of EGFP fluorescence profile 48 h post-transfection (A) and stable transfection (B). Cells with pIRES-EGFP-2 represented of low EGFP protein level. Cells with pIRES-EGFP-4 and pIRES-EGFP-6, represented of medium EGFP protein level. Cells with pIRES-EGFP-9 represented of high EGFP protein level.

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  Figure 4. Relative EGFP protein expression levels in CHO-S cells. Transient EGFP expression was measured by flow cytometry 48 h post-transfection. All the experiments were repeated three times. It indicates that the EGFP expression levels from pIRES-EGFP-9 with the IRES of HRV were higher compared with those from other vectors (P < 0.05).

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  Figure 5. Representative positive colony in stably transfected CHO-S cells. Transfection and drug selection were carried out as stated in the portion of materials and methods. Single clones growing from each of the population were photographed with fluorescence microscope. Three stably single clone pools were chosen for vectors. EGFP fluorescence in cells stably transfected with pIRES-EGFP-9 (A) pIRES-EGFP-8 (B) and pIRES-EGFP-2 (C). (D) Image of colony under white light. Scale bar, 50 μm.

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  Figure 6. Relative EGFP expression levels and copy numbers in stably transfected CHO-S cells. Cells were tested for the MFI with the FACS Calibur (A) and copy number was measured by qPCR (B). It indicated that relative EGFP protein levels in stable transformation cells was decreased and cells transfected with pIRES-EGFP-9 with the IRES of HRV had the highest gene copy numbers.

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  Figure 7. Analysis of EPO protein expression by western bolt. The vectors pIRES-EPO-8 and pIRES-EPO-9 were transformed into CHO-S cells, and four clones of cells were chosen, respectively. Lane 1–4, pIRES-EPO-9; Lane 5–8, pIRES-EPO-8.