Extended Data Fig. 6. Analysis of competitive repopulation experiments and lentiviral vector transduction.

(A) Schematic representation of lentiviral constructs used. (B) Representative example of transduction efficiency of purified HSCs with IRES-GFP and IRES-mCherry vectors 24–48h post-transduction. (C) Schematic representation of transduction and subsequent competitive repopulation experiments. (D) Representative flow cytometric plots of the gates used to analyze recipients of competitive repopulation experiments. The CD45.1/CD45.2 gates were also applied to the myeloid, B and T cell gates to determine donor contribution to individual lineages. In this example, HSCs had been transduced with an IRES-GPF lentiviral vectors (efficiency 80%, see (B)). Although donor repopulation was high in the periphery (upper left panel, 71.8%) no GFP was detected (lower left panel), suggesting silencing of the vectors. (E) Quantification of proviral copy number in donor-derived (CD45.2⁺) 15 weeks after transduction and competitive transplantation of HSCs. Despite silencing of the vector (see (D)), approximately 1 proviral copy was present per donor HSC-derived cell. n=3 biological replicates; *P<0.05; two-tailed student’s t test. (F) Representative example of transduction efficiency of purified HSCs with IRES-GFP and Mfn2-IRES-GFP lentiviral vectors. (G) Expression of Mfn2 mRNA in CD150^hi and CD150^lo HSCs 24h post-transduction relative to EV control (left panel) and in donor-derived HSCs, CMPs and lineage⁺ cells 15 weeks after competitive transplantation of 200 transduced HSCs together with 2×10⁵ CD45.1⁺ competitor cells (right panel). The data indicate partial silencing of the vector in HSCs, and complete silencing in the progenitors and more mature cells. n=3 biological replicates; *P<0.05; two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test. (H) Donor (CD45.2⁺) chimerism 15 weeks after transplantation of 200 CD45.2⁺ HSCs transduced for 24h with SIN LTR constructs pLVX-IRES-GFP or pLVX-Mfn2-IRES-GFP together with 2×10⁵ CD45.1⁺ competitor fetal liver cells into lethally irradiated CD45.1⁺CD45.2⁺ recipients mice. Plots, mean±s.e.m.; n≥19 recipients from four transplantation experiments; *P<0.05; two-tailed student’s t test. (I) Same experiments as in (H), but using the non-SIN LTR vector, pHR. Plots, mean±s.e.m.; n=5 recipients; *P<0.05; two-tailed student’s t test.