(A) Detection of the transduced lymphocytes in vivo. Genomic DNA was isolated from the PBMC from RQ876 following the second infusion and RQ900 and RQ1114 following the third infusion of transduced cells were analyzed semiquantitative PCR for the Neo^R gene. The samples labeled Pre represent the percentage of gene marked cells present in the animal immediately before reinfusion of the transduced lymphocytes. All samples were normalized to PCR reactions performed using the β-actin gene. The percentage of Neo^R(+) cells in the standard curve were 10%, 1%, 0.1%, and 0.01%. (B) Neo^R marking of fractionated PBL. PBL were isolated from RQ876 120 days following the second infusion of transduced CD4-enriched lymphocytes, and from RQ816, a negative control animal. The PBMC were sorted into CD4⁺ and CD8⁺ cell populations by fluorescence activated cell sorting (FACS). The purity of the separated cell subpopulations used for genomic DNA isolation exceeded 97%. Direct PCR with [³²P]-dCTP (deoxycytosine triphosphate) was performed for the detection of the Neo^R gene. The standard curve for the PCR was generated by isolating genomic DNA from a G418 resistant population of LSGN vector (glucocerebrosidase/Neo^R vector) transduced cells. The genomic DNA was diluted 1:1, 1:5, 1:25, 1:125, and 1:625 for preparation of the standard curve. (C) Trafficking of the transduced CD4 lymphocytes to the LNs. At various times following the second reinfusion of transduced cells (indicated as D-21, -100, or -87), inguinal LN biopsies and PB were isolated from all of the animals. Genomic DNA was isolated from the samples and used for direct PCR for the Neo^R gene in the presence of [³²P]-dCTP. The Neo^R(+) standards of 10%, 1%, 0.1%, and 0.01% are shown.