Functional analysis of various promoters in lentiviral vectors at different stages of in vitro differentiation of mouse embryonic stem cells

Abstract

Given the therapeutic potential offered by embryonic stem (ES) cells, it is critical to optimize stable gene delivery and expression at different developmental stages of ES cell differentiation. Here, we systematically analyzed lentiviral vectors containing the following promoters: the human elongation factor 1alpha (EF1alpha) promoter, the human cytomegalovirus (CMV) immediate early region enhancer-promoter, the composite CAG promoter (consisting of the CMV immediate early enhancer and the chicken beta-actin promoter), the human phosphoglycerate kinase 1 (PGK) promoter, the murine stem cell virus (MSCV) long terminal repeat (LTR), or the gibbon ape leukemia virus (GALV) LTR. Our results show that the EF1alpha promoter directed robust transgene expression at every stage of mouse ES cell differentiation, whereas the CMV promoter drove transgene expression only during late stages. Similarly, the CAG and PGK promoters drove transgene expression at a significant level only during late stages. The MSCV LTR and the GALV LTR exhibited much lower promoter activities at all stages. Interestingly, mouse ES cells transduced with the EF1alpha promoter-containing lentiviral vector lost most of their transgene expression during in vitro differentiation to neural precursors and neuronal cells. Our results demonstrate that different cellular and viral promoters exhibit very distinct and dynamic properties not only in terms of promoter strength but also with respect to differentiation stage-specific activity.

Figure 1. Transduction efficiency and cytotoxicity of lentiviral vectors at different stages—embryonic stem (ES) cells, embryoid bodies (EBs), and neural precursors—of in vitro ES cell differentiation

(a) Transduction efficiency and (b) cytotoxicity were determined using the SIN-EF1α-GFP-W lentiviral vector and multiplicities of infection ranging from 1 to 10. For transduction efficiency, the percentage of green fluorescent protein (GFP)⁺ cells 72 hours after transduction was analyzed by fluorescence-activated cell sorting. To measure the cytotoxicity of vesicular stomatitis virus G-pseudotyped lentiviral vectors, we assessed cell viability by trypan blue exclusion 72 hours after transduction. Each determination was performed in duplicate. Similar results were observed from three independent experiments.

Figure 2. Activity of each promoter at stage 1 (undifferentiated embryonic stem cells after fluorescence-activated cell sorting)

(a) Histograms showing green fluorescent protein (GFP) fluorescence 72 hours after transduction. (b) Pairwise comparisons of values of GFP fluorescence for corresponding vectors. Similar results were obtained in a second independent experiment. CMV, cytomegalovirus; EF1α, elongation factor 1α; GALV, gibbon ape leukemia virus; LTR, long terminal repeat; MSCV, murine stem cell virus; PGK, phosphoglycerate kinase 1.

Figure 3. Activity of each promoter at stage 2: embryoid bodies formed from fluorescence-activated cell sorted (FACS) green fluorescent protein (GFP)⁺ embryonic stem (ES) cells

Transduced GFP⁺ J1 ES cells with each lentiviral vector were isolated by FACS, in vitro differentiated to embryoid bodies, and then trypsinized to single cells using 0.25% trypsin-EDTA. The average level of GFP expression was determined by flow cytometric analysis after 10 days′ transduction. (a) Histograms showing GFP fluorescence 3 days after transduction. (b) Pairwise comparisons of values of GFP fluorescence for corresponding vectors. Similar results were obtained in a second, independent experiment. CMV, cytomegalovirus; EF1α, elongation factor 1α; GALV, gibbon ape leukemia virus; LTR, long terminal repeat; MSCV, murine stem cell virus; PGK, phosphoglycerate kinase 1.

Figure 4. Activity of each promoter at stage 4 (neural precusors) of in vitro embryonic stem (ES) cell differentiation

(a) Examination of mean fluorescence intensity of green fluorescent protein (GFP) expression by fluorescence-activated cell sorting analysis. Similar results were obtained in a second, independent experiment. (b) Analysis of GFP expression using fluorescence microscopy. Three days after transduction, cells were fixed, mounted, and analyzed for GFP expression using fluorescence microscopy. (c) Quantitation of GFP messenger RNA (mRNA) from stably integrated lentiviral vectors by real-time polymerase chain reaction analysis. ES cells were differentiated into embryoid bodies, and this was followed by further selection of neural precursors as described in Materials and Methods. Neural precursor cells were transduced with each lentiviral vector. CMV, cytomegalovirus; DAPI, 4′,6-diamidino-2-phenylindole; EF1α, elongation factor 1α; GALV, gibbon ape leukemia virus; LTR, long terminal repeat; MSCV, murine stem cell virus; PGK, phosphoglycerate kinase 1.

Figure 5. Activity of each promoter at stage 5—embryonic stem (ES) cell-derived neurons—of in vitro ES cell differentiation

(a) Analysis of green fluorescent protein (GFP) expression using fluorescence microscopy. Three days after transduction, cells were fixed, mounted, and analyzed for GFP expression using fluorescence microscopy. (b) Quantitation of GFP messenger RNA (mRNA) from stably integrated lentiviral vectors by real-time polymerase chain reaction analysis. ES cells were differentiated into embryoid bodies, and this was followed by further selection and expansion of neural precursors as described in Materials and Methods. ES cell-derived neurons were infected with each lentiviral vector. CMV, cytomegalovirus; DAPI, 4′,6-diamidino-2-phenylindole; EF1α, elongation factor 1α; GALV, gibbon ape leukemia virus; LTR, long terminal repeat; MSCV, murine stem cell virus; PGK, phosphoglycerate kinase 1.

Figure 6. Diagram indicating the distinct and dynamic transcriptional activities of individual promoters

For relative comparison, the promoter activity of the strongest promoter was set at 1.0 at each stage and the relative values of other promoters are shown. Promoter activities are based on fluorescence-activated cell sorting analysis at the ES, EB, and NP stages and real-time polymerase chain reaction analysis at the FD stage. CMV, cytomegalovirus; EB, embryoid body; EF1α, elongation factor 1α; ES, undifferentiated embryonic stem cell; FD, fully differentiated cell; GALV, gibbon ape leukemia virus; MSCV, murine stem cell virus; NP, neural precursor; PGK, phosphoglycerate kinase 1.

Figure 7. Stable expression and transcriptional silencing of transgene after transduction by a lentiviral vector

(a) Green fluorescent protein (GFP)⁺ cells sorted 3 days after transduction were cultured for 60 days and analyzed by fluorescence microscopy at days 3 and 60. The experiment was repeated twice, and percentages of GFP⁺ cells were determined. (b) DIC (bottom) and fluorescence microscopy (upper) images of mouse embryonic stem (mES) cell colonies after transduction. (c) Transgene expression during in vitro differentiation of transduced mES cells. Shown are the microscopy images of transduced mES cells that were cultured for 28 days without passage. (d) Cells of each stage were fixed, mounted, and analyzed for GFP expression using fluorescence microscopy. Phase (upper) and fluorescence microscopy (bottom) images in mES cell and embryoid body. DAPI (4′,6-diamidino-2-phenylindole) counterstain in neural precursors (NPs) and fully differentiated (FD) cells was used for visualization (DAPI images, upper; fluorescence microscopy images, bottom). ES, undifferentiated ES cells; EB, embryoid body; FD, fully differentiated cell; NP, neural precursor. (e) Total RNAs were isolated from cells of each stage during in vitro differentiation of the elongation factor 1α (EF1α)-transduced mES cells and then quantitative real-time polymerase chain reaction analysis was performed. Relative expression of messenger RNAs (mRNAs) was assessed by normalizing levels of complementary DNA to the signal from glyceraldehyde-3-phosphate dehydrogenase mRNA.