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. 2016 Sep 17;94(11):1152–1168. doi: 10.1002/jnr.23847

Figure 1.

Figure 1

Novel lentiviral vectors support erythroid‐ and myeloid‐specific transgene expression. A: Novel self‐inactivating (SIN) vectors carrying erythroid (IHK)‐ and myeloid (146gp91)‐specific promoters. ssp, Synthetic secretory signal peptide; psp, parental secretory signal peptide; co‐GALC, codon optimized mouse GALC cDNA; myc, myc tag; 146gp91, myeloid‐specific promoter; IHK, erythroid/megakaryocyte‐specific promoter; IRES, internal ribosome entry site; WP, woodchuck hepatitis virus posttranscriptional regulatory element; ΔU3 LTR, Self‐inactivating (SIN) LTR deleted of the parental enhancer promoter. B: Lentiviral vectors carrying the IHK promoter support erythroid‐specific transgene expression in vitro. FACscan analysis of GFP expression in mouse erythroleukemia (MEL) cells, human 293T cells, mouse Lin– cells, and mouse Lin–Sca1+Kit+ (LSK) cells following transduction with lentiviral vectors carrying either the IHK promoter (pTK1580 and pTK1582) or the CMV promoter (pTK945). MEL and 293T cells were analyzed either before or after HMBA‐induced erythroid differentiation. Untransduced MEL and 293T cells served as controls. Percentage of GFP‐positive cells is shown. Mean fluorescence intensity (MFI) presents levels of GFP expression. Note that GFP expression from IHK‐containing lentiviral vectors was detected only in HMBA‐induced MEL cells. C,D: Lentiviral vectors carrying the 146gp91 promoter support myeloid‐specific transgene expression in vitro. C: Lentiviral vectors carrying the GFP reporter gene under control of either the myeloid 146gp91 promoter (pTK1607) or the CMV promoter (pTK945) were employed to transduce human 293T cells, cells of the human THP‐1 monocyte cell line, the above‐mentioned mouse LSK cells, and mouse BM cells expressing the macrophage surface marker Mac1+. FACScan analysis of GFP expression was employed as described for B. Note that high levels GFP expression from the CMV promoter were detected in all target cells excluding the Thp1 cells. CMV‐regulated expression was higher in LSK than in Mac1+ cells. On the other hand, GFP expression driven by146gp91 was high in THP1 and very low in 293T cells. Furthermore GFP expression level in Mac1+ cells was significantly higher than the level of expression detected in LSK cells. D: Lentiviral vectors carrying the firefly luciferase under control of either a myeloid promoter (pTK1607) or a CMV promoter (pTK993) transduced 293T and THP‐1 cells. Luciferase activity in relative light unit (RLU) was normalized per milligram protein and VCN per cell. Luciferase activity generated by each vector in 293T served as a reference baseline. The ratio or fold expression of luciferase activity from these vectors in THP1 cells relative to luciferase expression in 293T cells was calculated. Note that CMV expression in THP‐1 cell was dramatically lower than its expression in 293T cells. On the other hand, luciferase expression per vector genome from the myeloid promoter increased 33‐fold relative to its expression in 293T cells.