Figure 3.

Incorporation of miR-106b target sequence (miRT) into the 3′-UTR of EGFP effectively detargets lentiviral-mediated expression in proliferating basal cells. (A) Diagram of the bidirectional promoter proviral lentiviral plasmids (pLV-dt/EGFP-miRT and pLV-dt/EGFP-RmiRT) used to generate lentivirus and test detargeting in basal cells. The box legend to the right highlights the components of these proviral plasmids as described in detail within the Figure 2A legend. LV-dt/EGFP-miRT is the experimental vector harboring a CBA promoter driven nuclear targeted EGFP (EGFP-nls) with miR-106b target sequence (4× miR target or mirT) in the reverse orientation. In the forward direction, the PGK promoter drives expression of the Tomato reporter, which is unaffected by miR-106b. LV-dt/EGFP-RmiRT is a control vector with the miRNA target sequence in the reverse orientation. (B) LV-dt/EGFP-miRT and LV-dt/EGFP-RmiRT viruses were used to transduce primary human airway basal cell in SAGM-EA cultures. The Tomato-positive (red) cells indicate the virally transduced cells. EGFP expression is seen in dt/EGFP-RmiRT control transduced cells but not in cells transduced with the detargeted LV-dt/EGFP-miRT vector. Scale bar, 100 µm. (C) Basal cells transduced with LV-dt/EGFP-miRT vector, and FACS isolated for Tomato-positive cells, were transfected with miRT-106b inhibitor sequences to block detargeting or mock transfected. Scale bar, 100 µm. (D) The cells in (B and C) were analyzed by flow cytometer and are shown in dot plots to the right of the corresponding images for each condition. The percentage of cells are indicated in each quadrant: Q1 (Tomato-positive only cells), Q2 (Tomato and EGFP double-positive cells), Q3 (EGFP-positive only cells), and Q4 (non-fluorescent cells).