Relationship between the number of transplanted HSCs and detected barcodes. (A) Overview of limiting dilution experiments. LSK48⁻150⁺ cells were purified by cell sorting. For establishing the frequency of functional HSCs in this population, naïve cells were transplanted into irradiated recipients. Alternatively, LSK48⁻150⁺ cells were transduced with barcoded viruses and different doses of transduced cells were transplanted into irradiated hosts, either without (middle arrow) or with (right arrow) selection for GFP⁺. Mouse strains, competitors, and irradiation regimen used in every experiment are indicated. Congenic donor and recipient B6 animals were used to allow donor and recipient cell discrimination. Granulocytes (G), B- and T-lymphocytes (indicated as B and T, respectively) were isolated for further barcode analysis at regular time points after transplant. (B) Assessment of HSCs frequencies by limiting dilution analysis in the naive LSK48⁻150⁺ population, in transduced nonsorted and in sorted GFP⁺ cells. (C) Relationship between the expected number of transplanted HSCs and the number of barcodes, as detected in granulocytes. Each dot represents an individual mouse. Light gray circles indicate data generated using nonsorted cells and dark gray squares reflect experiments with sorted cells. In one mouse, data on granulocytes were not available for week 20, so week 28 data are shown instead with a black square. The best-fit line (line equation Y = 0.61X + 2.58) and 95% confidence intervals are plotted. Note that the 95% confidence interval includes X/Y intercept. (D) Observed and expected vector copy number per transduced cell at different transduction efficiencies. The average number of barcodes in 15 to 22 colonies is depicted as a function of gene transduction efficiency. (E) Same as shown in (C), but now data are shown for T-cell clones. Equation for best-fit line was Y = 0.36X + 8.5.