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Regulation of F8 promoter activity
moter length. Specifically, in ECV-304, where Ets-1 is expressed, we observed a 3- to 6-fold increase in activity of each pF8 using the sgRNA F8.2, while no significant changes were observed with the sgRNA F8.1 (Figure 4B). In HEK293T cells, however, where Ets-1 is insignificant, we observed an upregulation in pF8 activity, ranging from a 20- to 40-fold, likely explained by the lack of competi- tion for the BS involved (Figure 4C). Taken together, our results, highlight a central role of the sgRNA F8.2 in pF8 transactivation confirming the powerful role of the E2 site in promoter regulation.
In vivo maintenance of high endothelial specificity by shortened promoters
In order to assess the activity and specificity of the newly identified shortened pF8 sequences in vivo, we gen- erated LV expressing GFP under the control of pF8-600, pF8-446 or pF8-342, and we administrated 5x108 TU to C57BL/6 mice. pF8-1175 was used as a control. Two weeks after injection, hepatic GFP expression was detect- ed primarily in endothelial Lyve-1+ cells in all mice (Figure 5A), with rare F4/80+ macrophages GFP positive (GFP+) seen with the pF8-446 promoter (Figure 5B). Co-staining
Figure 6. Green fluorescent protein expression in spleens under the control of pF8 shortened promoter sequences. Representative immunofluorescence images from mice spleens, 2 weeks following the delivery of lentiviral vector (LV) carrying different promoters. In red the endothelial marker CD31 (left side) or the macrophage marker F4/80 (right side) with green fluorescent protein (GFP) (green). Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 100 mM. n=3-5 (mice); sections=5-7 per mouse.
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