Making V(D)J rearrangement visible: quantification of recombination efficiency in real time at the single cell level.
V(D)J recombination is of fundamental importance for the diversity of immunoglobulin and T cell receptor genes. An enhanced green fluorescent protein (EGFP) based assay was successfully developed to monitor V(D)J recombination efficiency. This assay makes V(D)J recombination visible at the single cell level in real time. Surprisingly, despite a high (60% to 90%) transfection efficiency, the EGFP based V(D)J recombination efficiency was found to be low ( approximately 1%) in 293 cells. The EGFP based V(D)J recombination efficiency correlated well with that achieved by the classical V(D)J recombination assay. The EGFP based V(D)J recombination efficiency depended on the relative RAG (recombination activating gene)-1 and RAG-2 but not Artemis expression vector concentrations used for co-transfection. A rise of RAG-1 dosage increased recombination efficiency. In contrast, a surplus of RAG-2 inhibited V(D)J recombination efficiency. The test differentiates RAG null mutants as seen in human severe combined immunodeficiency (SCID).
Zheng
Xiao
X
Department of Transfusion Medicine, University Hospital Ulm, Institute for Clinical Transfusion Medicine and Immunogenetics, German Red Cross Blood Service Baden-Württemberg-Hessen, Institute Ulm, Helmholtzstrasse 10, D-89081 Ulm, Germany.
Schwarz
Klaus
K
D001483:Base Sequence; D002460:Cell Line; D003199:Computer Systems; D004268:DNA-Binding Proteins / Q000235:genetics*; D005434:Flow Cytometry; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D049452:Green Fluorescent Proteins / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D007158:Immunologic Techniques; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D011995:Recombination, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; C499311:RAG2 protein, human; C485184:enhanced green fluorescent protein; C064658:RAG-1 protein; D049452:Green Fluorescent Proteins