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. 2014 Jul 14;3(9):1032–1042. doi: 10.5966/sctm.2014-0011

Figure 2.

Figure 2.

Validation of lentiviral vector. (A): Representation of the human FOXA2 gene with the promoter region (in red) and the 5′-untranslated region (in orange) used to regulate the expression of GFP (see also supplemental online Fig. 4) and later removed to generate a promoterless vector (pLESS.GFP). (B): Quantification of GFPpos cells (%) and mean fluorescence intensity by flow cytometry, 3 days after transduction of HepG2 cells, a human cell line with robust constitutive expression of FOXA2. Excision of the promoter sequence from the vector demonstrates the specificity of the construct, at low MOI; at high MOIs, it showed leakage with increase in the percentage of GFPpos cells, although the mean intensity was always very low. (C, D): Transduction of human neural progenitors was carried out at approximately DIV28 with sustained expression of GFP in the neural cultures. Shown are representative microphotographs of live human embryonic stem cell-derived neurons 1 week after lentiviral transduction. (E, F): IF and confocal analysis confirmed that GFP expression was restricted to FOXA2pos cells and was colocalized with neuronal markers like NCAM and βIII tubulin. (G): GFPpos neurons expressed Tau and TH and displayed mature features with axonal localization of Tau. Inset (g) shows a higher magnification of the boxed area in (G). (H): Transduction with this vector did not modify the endogenous levels of FOXA2 transcript or the expression of other relevant genes by quantitative real-time polymerase chain reaction, 1 week post-transduction. Scale bars = 50 μm in (E) and (F) and 100 μm in (D) and (G). Abbreviations: cPPT, central polypurine tract; CTRL, control; DANs, dopamine neurons; DAPs, dopamine progenitors; DIV, day in vitro; eGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; hESC, human embryonic stem cell; IF, immunofluorescence; LTR, long terminal repeat; MOI, multiplicity of infection; NCAM, neural cell adhesion molecule; NPs, neural progenitors; tdx, transduction; UTR, untranslated region; wk, week; WPRE, Woodchuck hepatitis virus Postranscriptional Regulatory Element.