Tet-inducible system for postmitotic LCs. (A) Immature CD34⁺-cell–derived LC clusters were generated under LC-promoting conditions and analyzed by FACS for CD1a, Langerin, and CD11b expression (numbers depict percentages). Photomicrograph shows typical round LC clusters. (B) Schematic representation of bicistronic retroviral vectors. The first (TA-mCD8α) encodes the tet-activator (TA) upstream of IRES-mCD8α; the second is a self-inactivating vector that encodes a tet-response element (TRE) followed by either IRES-GFP or IRES-NGFR (HR-GFP/NGFR). (C) CD34⁺ cells are sequentially infected with the constructs shown in panel B and cultured in a 2-step differentiation model as indicated. Gene expression is turned on by DOX (2 μg/mL) in immature LCs on day 8 and analyzed 24 hours or 48 hours later. (D) Representative FACS diagrams show day-10 LCs grown with or without DOX. Induced gene expression is marked by GFP expression.