Figure 2

Cell-type specificity of recombination. (a) Recombination activating gene (RAG)1 and RAG2 expression in pre-B 300-19P cells (REC+) and WEHI231 B cells (REC-) analysed by reverse transcription polymerase chain reaction (RT-PCR). Actin transcripts were detected in both cell lines. The RAG genes are intronless and assays were performed using reverse transcriptase (RT+) as well as in its absence (RT-) to ensure that signals were not derived from contaminating genomic DNA. (b) Detection of recombination by PCR (using the F1 and R1 primers shown in Figure 1). A PCR product of 155 bp was predicted following recombination. (c) RT-PCR to detect expression of EGFP mRNA from the transfected substrates. The PCR product diagnostic of EGFP transcripts from the recombined substrate is 155 bp. The 975-bp product detected from the deletion substrate transcripts represents a mRNA that traverses the entire DsRed gene and continues on through the EGFP gene. No products were detected in the absence of reverse transcriptase (- RT enzyme). Both DNA recombination and mRNA expression assays were performed from untransfected cells (Untrans) and in cells stably transfected with the deletion and inversion substrates (non-clonal populations grown under continuous selection) using 300-19P (REC+) and WEHI231 (REC-) cells. A (-) symbol indicates no added template.