Our strategy to generate stable ffluc⁺ primary ALL samples. Bulk transduction of primary patient ALL, freshly obtained from NSG mouse bone marrow, via lentivirus results in low-level GFP/ffluc expression, which can be purified by flow sorting. Three primary patient ALL samples (ALL-1, ALL-2, and ALL-3) were harvested from moribund NSG mice, transduced overnight, and viable cells injected into primary recipients via tail vein. All 3 samples engrafted with a small GFP⁺ population, which was then purified using the gating strategy shown (sample ALL-1 sorting is shown). This enrichment results in stable, > 99% bioluminescent primary ALL with resultant early detection and evenly disseminated disease in secondary (shown in bottom panels) and later recipient mice.