Experimental strategy.

(A) Retrovirus constructs were generated by subcloning cDNA for wild-type SOCS-1 or mutated SOCS-1 lacking a functional SH2 domain (SOCS-1:SH2*) into the MIEV retroviral vector. The presence of an internal ribosomal entry site (IRES) upstream of a GFPreporter gene leads to the production of bicistronic mRNA under the control of the 5′ long terminal repeat (LTR). As control, a retroviral vector was generated containing only the Pcmvpromoter derived from the pcDNA3.1 plasmid. (B) Experimental procedure for retroviral gene transfer into fetal liver–derived hematopoietic precursors (top) or fetal thymic lobes (bottom). Hematopoietic precursors were enriched from fetal liver by complement-mediated depletion of CD24⁺ cells. Precursors were then cocultured on a monolayer of GP+E 86 retroviral packaging cells. The following day, infected cells were sorted based on the coexpression of GFP and CD117 and subsequently introduced into dGuo-treated fetal thymic lobes to allow T-cell differentiation to occur. Alternatively, fetal thymic lobes were cocultured with GP+E 86 cells and subsequently cultured under normal FTOC conditions.