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R. Famà et al.
sanalysis, these sgRNA guides, F8.1 and F8.2, mediate the recruitment of deactivated Cas9 fused to a tripartite tran- scriptional activator (VPR)34 to the two promoter regions encompassing E3 (-313 to -319) and E2 (-219 to -223) Ets- 1 BS, respectively (Figure 4A). In order to further demon- strate the central role of E2 in the modulation of pF8 activ- ity, we transfected both ECV-304 and HEK293T cells with CRISPRa system using sgRNA F8.1 and F8.2 as reference
guides covering the essential E2 and the E3 Ets-1 BS. As a negative control guide we performed the same experi- ment using a sgRNA targeting coagulation Factor VII pro- moter (pF7) (Online Supplementary Figure S3). By applying this approach to pF8-1175, pF8-600 and pF8-446, we con- firmed the ability of CRISPRa to upregulate the promoter activity. Importantly, the efficiency of the system was dependent on the cell type, the sgRNA used and the pro-
A
B
Figure 5. F8 shortened promoters drive green fluorescent protein expression in hepatic endothelial cells. Immunofluorescence analyses of C57BL/6 livers 2 weeks after lentiviral vector (LV) LV.pF8-600.GFP, LV.pF8-446.GFP or LV.pF8.342.GFP delivery, compared to the full length LV.pF8-1175.GFP. (A) Representative pictures of liver sections stained for the endothelial marker (Lyve-1, in red) and the green fluorescent protein (GFP) using 400x magnification. (B) Liver of LV-injected mice dec- orated with the macrophage marker F4/80 (red) in combination with GFP (green). Scale bars, 50 mM. Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). n=3-5 (mice); sections=3-6 per mouse. (for antibodies specifications refers to the Online Supplementary Appendix).
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haematologica | 2021; 106(6)