Analysis of different promoter systems for efficient transgene expression in mouse embryonic stem cell lines

Abstract

Mouse embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and have the developmental capacity to generate all cell types of the body. Combined with efficient genetic manipulation and in vitro differentiation procedures, ES cells are a useful system for the molecular analysis of developmental pathways. We analyzed and compared the transcriptional activities of a cellular polypeptide chain elongation factor 1 alpha (EF), a cellular-virus hybrid (cytomegalo-virus [CMV] immediate early enhancer fused to chicken beta-actin [CBA]), and a viral CMV promoter system in two ES cell lines. When transiently transfected, the EF and CBA promoters robustly drove reporter gene expression, while the CMV promoter was inactive. We also demonstrated that the EF and CBA promoters effectively drove gene expression in different stages of cell development: naïve ES cells, embryoid bodies (EBs), and neuronal precursor cells. In contrast, the CMV promoter did not have transcriptional activity in either ES cells or EB but had significant activity once ES cells differentiated into neuronal precursors. Our data show that individual promoters have different abilities to express reporter gene expression in the ES and other cell types tested.

Figure 1. Plasmid map of pIRES/hrGFP, which was used for the expression of hrGFP under the control of three different promoter candidates CBA, CMV, and EF subcloned into the promoter site (for details see Materials and Methods)

MCS = multiple cloning site; HA = hemagglutinin tag; IRES = internal ribosome entry site.

Figure 2. Activities of the EF, CBA, and CMV promoters in D3 and J1 ES and 293T cells

Each hrGFP-expressing plasmid driven by one of the three different promoters was transiently transfected into the cell lines described. Thirty-six hours after transfection, cells were fixed, mounted, and analyzed for GFP expression using fluorescence microscopy.

Figure 3. Quantification of promoter strength in D3 and J1 ES and 293T cells

Each cell line was transfected with the individual hrGFP-expressing construct along with the luciferase-expressing plasmid pGL3 to control for transfection efficiency. The pGL3 vector drove reasonable levels of luciferase expression under the control of SV40 promoter in all cell lines transfected. Thirty-six hours after transfection, cell lysates were prepared and assayed for GFP expression and luciferase activity. Levels of GFP expression were normalized to the luciferase activity, and the relative units were calculated with the activity of the CBA promoter set to 100. Each promoter data set represents n = 4 from two independent experiments in the case of D3 and J1 cells, and n = 3 for 293T cells. Analysis of variance results were: for D3 cells, F_3,11 = 96.257, p <0.005; for J1 cells, F_3,11 = 62.972, p <0.005; for 293T cells, F_3,7 = 48.117, p <0.005. Post hoc analysis was performed with a significance level of 5%. * indicates that the activity of EF was significantly greater than that of CBA and CMV. # indicates that the activity of CBA was significantly greater than that of CMV. § indicates that the activity of CMV was significantly greater than that of CBA.

Figure 4. Activity of each promoter at different stages of in vitro ES cell differentiation

ES cells were differentiated into EB followed by further selection of neuronal precursors as described elsewhere [21] and in Materials and Methods. Undifferentiated ES, EB, and neuronal precursor cells were transfected with each of the hrGFP-expressing constructs driven by one of the three promoters (CBA, CMV, and EF). Thirty-six hours after transfection, cells were fixed, mounted, and analyzed for GFP expression using fluorescence microscopy.

Figure 5. Quantification of promoter strength in EB cells

Thirty-six hours after transfection, cell lysates were prepared and assayed for GFP expression and luciferase activity. Levels of GFP expression were normalized to the luciferase activity, and the relative units were calculated with the activity of the CBA promoter set to 100. Each promoter data set represents n = 3. The result of the analysis of variance was F_3,7 = 13.148, p <0.005. Post hoc analysis was performed with significance level of 5%, and * indicates that the activity of EF was significantly greater than that of CBA and CMV.