MEF and B cell manipulation for CSR

Wild-type, BRCA1^Δ11/Δ11, 53BP1^−/− and BRCA1^Δ11/Δ1153BP1^−/− mouse embryonic fibroblasts (MEFs) have been described (10 (link)). Resting B cells were isolated from spleen using anti-CD43 microbeads (Miltenyi Biotec) and stimulated to proliferate with 25 μg/ml LPS, 5 ng/ml IL-4 (both Sigma-Aldrich) and 0.5 μg/ml RP105 (BD PharMingen) (10 (link)). To overexpress RNF168, constructs encoding human RNF168^WT or RNF168^R57D (9 (link)) were subcloned into the pMX-PIE-IRES-GFP retroviral vector and used to transfect BOSC23 cells along with the pCL-Eco helper virus. Retroviral supernatant was collected 40–48 h later for infection of MEFs and B cells as previously described (38 (link)). pMX-PIE-based retroviruses encoding 53BP1^DB and 53BP1^DN have been described (11 (link),28 (link)). Class switch recombination was assayed on days 3 and 4 using biotinylated anti-IgG₁ and fluorochrome-conjugated anti-B220 antibodies (BD PharMingen).