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Making V(D)J rearrangement visible: quantification of recombination efficiency in real time at the single cell level

Xiao Zheng1, Klaus Schwarz

  • 1Department 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.

Journal of Immunological Methods
|August 29, 2006
PubMed

Insights

A new assay visualizes V(D)J recombination in real time. Researchers found low efficiency in 293 cells, dependent on RAG-1 and RAG-2 expression levels, aiding in identifying SCID mutations.

Area of Science:

  • Immunology
  • Molecular Biology
  • Genetics

Background:

  • V(D)J recombination is crucial for adaptive immune system diversity.
  • Generating diverse immunoglobulin and T cell receptors relies on V(D)J recombination.
  • Monitoring V(D)J recombination efficiency is essential for understanding immune development and disorders.

Purpose of the Study:

  • To develop and validate a novel assay for real-time, single-cell monitoring of V(D)J recombination efficiency.
  • To investigate the factors influencing V(D)J recombination efficiency in 293 cells using the new assay.
  • To assess the utility of the assay in identifying defects in V(D)J recombination, such as those found in severe combined immunodeficiency (SCID).

Main Methods:

  • Development of an enhanced green fluorescent protein (EGFP)-based assay to visualize V(D)J recombination.
  • Co-transfection of 293 cells with expression vectors for recombination activating gene (RAG)-1, RAG-2, and Artemis.
  • Quantitative analysis of EGFP expression to determine V(D)J recombination efficiency.
  • Comparison of the EGFP-based assay results with a classical V(D)J recombination assay.

Main Results:

  • The EGFP-based assay successfully visualized V(D)J recombination at the single-cell level in real time.
  • Despite high transfection efficiency, V(D)J recombination efficiency in 293 cells was approximately 1%.
  • Recombination efficiency correlated well with the classical assay and was dependent on the relative concentrations of RAG-1 and RAG-2 expression vectors.
  • Increased RAG-1 dosage enhanced recombination, while excess RAG-2 inhibited it.

Conclusions:

  • The EGFP-based assay is a reliable tool for monitoring V(D)J recombination efficiency in real time.
  • RAG-1 and RAG-2 expression levels critically influence V(D)J recombination efficiency.
  • This assay can effectively differentiate RAG-deficient mutants, relevant for diagnosing human SCID.

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