V(D)J recombination assay in RS-SCID fibroblasts withArtemis gene mutations.
(A) Schematic representation of the signal joint assay (sj) and the coding joint assay (cj) in which the recombination substrates pGG49 and pGG51 were used, respectively. Recombination signal sequences are depicted as triangles and the flanking coding sequence as rectangles. The signal joints are retained in the recombination substrate when pGG49 is used, while coding joints are retained in the recombination substrate when pGG51 is used. Signal joints and coding joints were detected with nested PCR analysis using a 32P-labeled primer (see “Materials and methods”). (B) Primary fibroblasts of Artemis-1 and Artemis-2 transfected with RAG1 andRAG2 expression constructs and the recombination substrate formed signal joints but no coding joints. FN1 control primary fibroblasts can form both signal joints and coding joints. Defective coding joint formation in Artemis-1 and Artemis-2 could be complemented by cotransfection of the wt Artemis expression construct (+Art). (C) SV40-immortalized fibroblasts of Artemis-3 were also able to form signal joints but not coding joints. Like in Artemis-1 and Artemis-2, coding joint formation occurred upon cotransfection of the wt Artemis expression construct. SV40-immortalized MCR5 fibroblasts were used as control.
