Retroviral Transduction of Pro-B Cells

Pro–B cells were isolated from tibias, femurs, and humeri of RAG2^−/−Myc^I/I mice at 4–10 wk of age by immunomagnetic enrichment with anti-B220 MicroBeads (Miltenyi Biotec). Cells were cultured at 2.0 × 10⁶ cells/ml in the presence of IL-7 (5 ng/ml; Sigma-Aldrich) in complete RPMI (RPMI-1640 supplemented with l-glutamine [Gibco], sodium pyruvate [Gibco], antibiotic/antimycotic [Gibco], Hepes [Gibco], 55 µM β-mercaptoethanol [Gibco], and 10% fetal calf serum [HyClone]). IL-7 was replenished on day 2. On days 3 and 4, cell supernatants were replaced with retroviral supernatants resulting from cotransfection (Fugene-6; Roche) of BOSC23 cells with pCL-Eco and pMX-I-SceI-P2A-RAG2^core-EGFP or pMX-I-SceI-EGFP plasmids 3 d before (Robbiani et al., 2008 (link)). Spinoculation was at 1,111 g for 1.5 h in the presence of 2.5 µg/ml polybrene, 5 ng/ml IL-7, and 20 mM Hepes. After 6–8 h at 37°C, on day 3, retroviral supernatants were replaced with original supernatants, whereas on day 4, cells were collected for IL-7 washout and replating in fresh complete RPMI. Cells were harvested after 2.5 d of IL-7 depletion, sorted for EGFP expression with a FACSAria instrument (BD), pelleted, and snap-frozen on dry ice. Samples infected with pMX-I-SceI-P2A-RAG2^core-EGFP are referred to as RAG2^core, and those infected with pMX-I-SceI-EGFP are referred to as RAG2^−/−.