Figure 6.

No RAG reexpression in RAG⁻ splenic B cells after in vitro stimulation. (A) Sorted B220^hiIgD⁺ splenic B cells (99% purity by FACS^® reanalysis with 0.1% contaminating B220^lo cells) from a pool of five 15-wk-old C3H mice were left uncultured or were cultured in vitro for 3 d in RPMI medium as indicated. RT-PCR analysis of the RAG1 transcript levels was performed using β-actin for normalization. Uncultured BM cell RNA served as a positive control. Absolute levels of RAG transcripts in the sorted B cell samples were ∼1:2,000 of those in total BM as determined by serial dilutions (data not shown). The functional integrity of these cultures was confirmed by their ability to perform isotype class switching 4–5 d after appropriate stimulation (data not shown). (B) Splenic B cells sorted based on very high B220 expression were cultured in vitro. RNA was purified from uncultured cells or from samples cultured for 2 d, and RAG1 and RAG2 transcripts were assayed by RT-PCR with high sensitivity (up to 45 cycles) using β-actin for normalization. Uncultured BM cells were analyzed as a positive control.