Induction of TCRDrecombination by E2A or HEB in the presence of RAG1 and RAG2.

Both E2A and HEB in combination with RAG1 and RAG2 have the ability to induce TCRD recombination in nonlymphoid cells. (A) Schematic diagram of the human TCRD/TCRA locus. Presented are the Vδ, Dδ, and Jδ gene segments that are positioned between the Vα and Jα-Cα regions in the TCRA locus. The δREC and ψJα gene segments that flank the TCRD locus are involved in deletion of the TCRD gene prior toTCRA recombinations. (B) (C) BOSC 23 cells were transfected with E47, HEB, RAG1, and RAG2 expression vectors. Following genomic DNA isolation, 200 ng DNA of the various transfectants was used for PCR amplification by means of Dδ2-5′ RO2 plus Dδ3-3′ N (panel B) or Vδ2-5′ plus Dδ3-3′ N (panel C) primers, which are used for specific detection of Dδ2-Dδ3 and Vδ2-Dδ3 rearrangements, respectively. Thymus DNA was used as positive control, and mock-transfected BOSC 23 as nonspecific template control. PCR products were run on a 2% agarose gel and stained with ethidium bromide. Rearranged Dδ2-Dδ3 and Vδ2-Dδ3 PCR products were observed upon combined transfection of either E2A or HEB together with RAG1 plus RAG2 (RR). Dδ2-Dδ3 PCR products were also detectable in genomic DNA derived from transfectants expressing RR only (lane RR), though at much lower levels as quantitated via RQ-PCR (see “Results”).