Abstract
The cells of the adaptive immune system, B and T lymphocytes, each generate a unique antigen receptor through V(D)J recombination of their immunoglobulin (Ig) and T cell receptor (TCR) loci, respectively. Such rearrangements join coding elements to form a coding joint and delete the intervening DNA as circular excision products containing the signal joint. These excision circles are stable structures that cannot replicate and have no function in the cell. Since the coding joint in the genome is replicated with each cell division, the ratio between coding joints and signal joints in a population of B cells can be used as a measure for proliferation. This chapter describes a real-time quantitative (RQ-)PCR-based approach to quantify proliferation through calculating the ratio between coding joints and signal joints of the frequently occurring intronRSS–Kde rearrangements in the IGK light chain locus. The approach is useful to study basic B-cell biology as well as abnormal proliferation in human diseases.
Key words
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- BCR:
-
B-cell antigen receptor
- D:
-
Diversity
- Ig:
-
Immunoglobulin
- IgH:
-
Ig heavy chain
- Igκ:
-
Ig kappa light chain
- Igλ:
-
Ig lambda light chain
- J:
-
Joining
- Kde:
-
Kappa-deleting element
- KREC:
-
Kappa-deleting recombination excision circle
- RAG:
-
Recombination activating gene
- RQ-PCR:
-
Real-time quantitative PCR
- RSS:
-
Recombination signal sequence
- V:
-
Variable
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Acknowledgments
MAB is supported by a Fellowship from the Ter Meulen Fund–Royal Netherlands Academy of Arts and Sciences. MCvZ is supported by an Erasmus University Rotterdam (EUR)-Fellowship, an Erasmus MC Fellowship, and by Veni grant 916.110.90 from ZonMW/NWO and by grant 689 of the Sophia Children’s Hospital Fund.
Conflict of Interest
JJMvD is inventor of the KREC assay, which has been patented (PCT/NL 2005/000761; priority date 25 Oct 2004) and licensed to InVivoScribe Technologies, San Diego, CA; revenues of the patent go to Erasmus MC. The other authors have declared that no conflict of interest exists.
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van Zelm, M.C., Berkowska, M.A., van Dongen, J.J.M. (2013). Studying the Replication History of Human B Lymphocytes by Real-Time Quantitative (RQ)-PCR. In: Küppers, R. (eds) Lymphoma. Methods in Molecular Biology, vol 971. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-269-8_6
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DOI: https://doi.org/10.1007/978-1-62703-269-8_6
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Publisher Name: Humana Press, Totowa, NJ
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