Differential bicistronic gene translation mediated by the internal ribosome entry site element of encephalomyocarditis virus

Fig. 1. Schematic diagrams of the mono- and bi-cistronic reporter vectors pEGFPIRESφ, pφIRESEGFP, pRlucIRESφ, pφIRESRluc, pRlucIRESFluc, pFlucIRESRluc, pIL2IRESφ, pφIRESIL2, pIL2IRESIL4, and pIL4IRESIL2. The plasmid pIRES, in which the multiple cloning sites (MCS) and IRES sequence are downstream of the immediate early promoter of cytomegalovirus (PCMVIE), was used as the vector backbone. The target genes encoding enhanced green fluorescent protein (EGFP) and Renilla luciferase (Rluc) were separated by the IRES element of encephalomyocarditis virus (EMCV) and expressed under the control of the CMV promoter. The intervening sequence (IVS) between PCMVIE and the MCS is an intron that is efficiently spliced out following transcription. SV40 polyadenylation signals downstream of the MCS direct the proper processing of the 3′ end of the mRNA of the gene of interest. Bacteriophage T7 and T3 promoters are located upstream and downstream of MCS A and B, respectively. pIRES contains the neomycin resistance gene (NeoR) to permit the selection of transformed cells.

Fig. 2. Comparison of EGFP expression from different constructs in mammalian cells. 3T3 cells were transfected with the indicated bicistronic vectors, including pEGFPIRESφ and pφIRESEGFP, by the liposome method. The EGFP expression in the cells was determined by fluorescence microscopy at 48 h post-transfection. The magnification is 100X.

Fig. 3. Real-time PCR evaluation of EGFP mRNA expression in cells transfected with the different IRES constructs. The RNA of 3T3 cells transfected with pEGFPIRESφ and pφIRESEGFP was isolated and reverse transcribed. Real-time PCR was performed to evaluate EGFP expression. The relative difference in EGFP cDNA levels was calculated and shown between the cells transfected with pEGFPIRESφ and pφIRESEGFP.

Fig. 4. Comparison of Rluc protein expression from different constructs in mammalian cells. 3T3 cells were transfected with the indicated mono- and bi-cistronic constructs by the liposome method. (A) Fluorescence activity generated in pIRES, pRlucIRESφ, pφIRESRluc, pRlucIRESFluc, and pFlucIRESRluc, (B) Ratios of Rluc expression by pRlucIRESφ, pφIRESRluc, and pRlucIRESFluc. (C) Ratios of Rluc expression by pφIRESRluc and pFlucIRESRluc The cells from cultures were harvested at 48 h post-transfection, and Rluc was measured by dual luciferase assay. (∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001).

Fig. 5. Comparison of IL-2 protein expression from different constructs in mammalian cells. 3T3 cells were transfected with the indicated mono- and bi-cistronic constructs by the liposome method. (A) IL2 generated in pIRES, pIL2IRESφ, pφIRESIL2, pIL2IRESIL4, and pIL4IRESIL2. (B) Ratios of IL-2 expression by pIL2IRESφ, pφIRESIL2, and pIL2IRESIL4. The cell culture media were harvested at 48 h post-transfection, and IL-2 was measured in the supernatants by ELISA. (∗: p < 0.05, ∗∗: p < 0.01).

Fig. 6. Differences of Flt3L protein expression from different constructs in mammalian cells. 3T3 cells were transfected with the indicated mono- and bi-cistronic constructs, including pFlt3LIRESφ and pFlt3LIRESBand3, by the liposome method. The cell culture media were harvested at 48 h post-transfection, and Flt3L was measured in the supernatants by ELISA. (∗∗∗: p < 0.001).