In vivo clone tracing by conventional PCR and limiting-dilution analysis.
(A) Conventional PCR. DNA isolated from FACS-purified granulocytes 11, 25, 42, 165, 339, and 528 days after transplantation of rhesus macaque 501 shows evidence for the presence of clones 3392, 2082, and 3397 (from left to right in each sample). The entire analysis from 16 time points is summarized in Figure 5A. M indicates molecular marker (100-bp ladder); C (PB), unrelated transduced peripheral blood control. (B) Limiting-dilution analysis. Note the presence of approximately equal copy numbers of clone 3392 in different lineages on days 240 (top lane) and 528 (bottom lane). Numbers indicate nanograms of DNA submitted to PCR analysis. Nd indicates not done; C, unrelated template control. (C) Total vector DNA. Per sample analyzed in Figures 2 and 3 (A, B), 10 ng DNA was analyzed for the presence of vector cDNA by semiquantitative PCR. Numbers indicate days after transplantation. G indicates DNA of purified peripheral blood granulocytes; MNC, purified peripheral blood mononuclear cells; T, T-cell DNA; B, B-cell DNA; DNA, transduced MNC DNA control; and H2O, no template control.
