Volume 15, Issue 9, Pages 1630-1639 (September 2007) Functional Analysis of Various Promoters in Lentiviral Vectors at Different Stages of In Vitro Differentiation of Mouse Embryonic Stem Cells  Sunghoi Hong, Dong-Youn Hwang, Soonsang Yoon, Ole Isacson, Ali Ramezani, Robert G Hawley, Kwang-Soo Kim  Molecular Therapy  Volume 15, Issue 9, Pages 1630-1639 (September 2007) DOI: 10.1038/sj.mt.6300251 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

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. Molecular Therapy 2007 15, 1630-1639DOI: (10.1038/sj.mt.6300251) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

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. Molecular Therapy 2007 15, 1630-1639DOI: (10.1038/sj.mt.6300251) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

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. Molecular Therapy 2007 15, 1630-1639DOI: (10.1038/sj.mt.6300251) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

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. Molecular Therapy 2007 15, 1630-1639DOI: (10.1038/sj.mt.6300251) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

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. Molecular Therapy 2007 15, 1630-1639DOI: (10.1038/sj.mt.6300251) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

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. Molecular Therapy 2007 15, 1630-1639DOI: (10.1038/sj.mt.6300251) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

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. Molecular Therapy 2007 15, 1630-1639DOI: (10.1038/sj.mt.6300251) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions