Regulation of Tcra gene assembly by a complex hierarchy of germline J α promoters

Tcra gene assembly is characterized by an orderly progression of primary and secondary V α to J α recombination events across the J α array, but the targeting mechanisms responsible for this progression are largely unknown. Previous studies revealed that the TEA promoter plays an important role in targeting primary Tcra rearrangements. We show that TEA and a novel promoter associated with J α 49 target primary recombination to discrete sets of C α -distal J α segments and together direct nearly all normal primary recombination events. Further, we show that TEA promoter deletion activates previously suppressed downstream promoters and stimulates primary rearrangement to centrally located J α segments. Central promoter derepression also occurs following primary rearrangement, thereby providing a mechanism to target secondary recombination events. The development of T lymphocytes bearing an αβ T cell receptor (TCR) depends on the somatic assembly of variable (V), diversity (D) and joining (J) gene segments by the process of V(D) J recombination 1 . Tcrb gene rearrangements occur first, in the CD4 - CD8 - double negative (DN) subset of thymocytes. Production of a functional TCR β protein then promotes differentiation to the CD4 + CD8 + double positive (DP) stage, during which Tcra gene rearrangements occur. Following expression of a cell surface αβ TCR, DP thymocytes are tested by positive selection to identify those with useful TCRs 2 . Only a small fraction of DP thymocytes are ultimately selected for further maturation. for germline transmission, or were retargeted for TEA deletion. The doubly targeted ES cells were screened by PCR and confirmed by Southern hybridization using the 3 ′ C δ probe in Kpn I digests. Doubly targeted ES cells were then treated to delete neo r in vitro or were used directly to produce chimeric mice that were bred to delete neo r in vivo . Δ TEA Δ J49 and Δ 5 ′ alleles were generated through both strategies. Gene targeted mice were subsequently bred with Rorc -/-28 , Rag2 -/- xTCR β transgene 29 and 129 mice. Breeding schemes insured that littermate controls always segregated wild-type 129 alleles. All mice were used in accordance with protocols approved by the Duke University Animal Care and Use Committee.