Table 2.

Methods for the Study of Receptor Editing

Method Description
cell culture systems. Bone marrow stromal pre-B cell cultures in which IL-7 is withdrawn will undergo sequential rearrangement of antibody L chains.
excision circles. Closed loops of DNA containing earlier Vκ-Jκ rearrangements can be detected in B cells that have undergone only a few cell divisions since L chain rearrangement. Sequence analysis can be used to evaluate editing precursors; see also reciprocal products
heavy chain allotype. Allelic differences in H chain constant regions can be used to generate antibodies that recognize H chains of particular mouse strains. These serological markers can be used to evaluate allelic inclusion and editing of H chains in mice that are heterozygous for two different H chain allotypes.
human Cκ heterozygous mice. This site-directed transgenic provides an allelic marker for the murine κ locus.
hybridomas and other transformed B cells. These monoclonal cell lines facilitate genetic analysis of reciprocal products from inversions and RS deleted alleles.
Ig transgenic and site-directed transgenic mouse models (see Table 3)
Jκ skewing. An increased usage of distal Jκ segments in highly edited B cell populations arises as a consequence of leapfrogging rearrangement at the κ L chain locus.
L chain isotype. Kappa (κ) and lambda (λ) L chains are the two L chain isotypes. Since the λ locus usually rearranges after the κ locus, B cells with more λ rearrangements have undergone more rounds of L chain gene rearrangement and may represent an edited population.
ligation-mediated PCR of DNA breaks. A technique that detects double strandedDNA breaks that are present transiently during the process of immunoglobulin gene rearrangement.
RAG transcript abundance or RAG reporter mice. RAG mRNA or protein expression in a particular B cell population or subset is consistent with ongoing immunoglobulin gene rearrangement.
reciprocal products. Vκ-Jκ rearrangements that occur by chromosomal DNA inversion create novel segments of DNA that may contain previous Vκ-Jκ rearrangements. This facilitates the analysis of editing precursors. See also excision circles.
RS deletion frequency. An increased frequency of RS rearrangement in B cells indicates more rounds of L chain gene rearrangement.
Sequence analysis. Ig gene sequence can identify VH replacement footprints. Single cell PCR amplification and sequencing of immunoglobulin genes can detect multiple productive rearrangements in a single cell.