Lymphoid reconstitution in γ_c-transduced mice.

(A) Flow cytometric analysis of peripheral blood T and B cells, using FITC-conjugated anti-TCRαβ and PE-conjugated anti-B220 antibodies. In these experiments, lymphocytes expressing T- or B-cell markers are calculated as a percentage of total nucleated cells to emphasize the kinetics of lymphoid reconstitution. (B-E) Analysis of γ_ctransgene integration and expression in γ_c-transduced animals. Polymerase chain reaction (PCR) was performed using exon 6- and exon 8-specific primers (B). The endogenous γ_c locus (0.8 kilobase [kb]) is amplified in wild-type mice but not in γ_c^- mice in which exon 6 has been deleted.16 γ_c-Transduced mice show a 0.4-kb product derived from the integrated γ_c transgene at 23 weeks postgraft. (C) Expression of the γ_c transgene was detected by RT-PCR from peripheral blood cells of control and γ_c-transduced animals at 7 weeks postgraft. Contaminating genomic DNA in the γc⁺ sample gives rise to a PCR product in the absence of reverse transcription. (D) Schematic of the retroviral construct used with the location of transmembrane (exon 6) and intracytoplasmic (exon 8) primers. (E) Expression of γ_c on γ_c⁺, γ_c^-, and γ_c-transduced cells. Staining of total thymocytes with isotype control monoclonal antibody (dotted line) and γ_c-specific monoclonal antibody (solid line) are shown.