Recombination activity of mutant RAG1. (A) Immunoblot analysis of Rag1^−/− pro-B Abelson cells expressing WT human RAG1, empty vector, or hypomorphic RAG1-mutant alleles from P1 (1428delC), P2 (1180C>T), and P3 (256_257delAA and 2164G>A). N-terminus–specific anti-RAG1 antibody (D36B3) cannot detect the N-terminus–truncated 256_257delAA. β-Actin serves as a loading control, and molecular weight markers (in kDa) are marked. (B) Flow cytometric analysis of Rag1^−/− pro-B Abelson cells containing a pMX-INV cassette with an inverted GFP sequence flanked by RSS (shown as gray triangles in [D]), and transduced with retroviral vectors expressing WT, empty, or hypomorphic RAG1 mutants. GFP⁺ events indicate rearrangement. (C) Recombination activity in transduced Rag1^−/− pro-B cells, expressed as a percentage of activity detected in cells expressing WT RAG1. For each construct, 3 independent experiments were performed, and mean value ± standard deviation is shown. (D) Schematic representation showing nonrearranged (NR) products flanked by Escherichia coli RV (EcoRV) (E) sites (5 kb), or EcoRV (E) and Nocardia corallina I (NcoI) (N) sites (2.2 kb). Recombination results in normal joining, producing a 3 kb fragment (coding joining [CJ]) flanked by N and E sites, or hybrid joining (HJ) flanked by V sites and producing a 4 kb fragment. Successful cleavage but unresolved CEs produce a 2 kb fragment. C4 represents the location of the probe to visualize the various fragments for Southern blotting. (E) Southern blot showing products of rearrangement of the pMX-INV vector in pro-B Abelson cells expressing ligase IV null, empty vector, WT RAG1, or one of the four hypomorphic RAG1 mutants. Hybridization with the C4 probe with EcoRV digest reveals NR, HJ, or CE events, and EcoRV+NcoI digest reveals HJ, CJ, or NR events. LTR, long terminal repeat; RCN, relative cell number; SJ, signal joining.