Two Models of Illegitimate V(D)J Recombination

<div><p>(A) V(D)J recombination generating an ESJ.</p> <p>(B) Ongoing V(D)J recombination of the ESJ with a RSS target in <i>trans,</i> leading to reintegration of the excised EC in the genome. The specific <i>trans</i>-CSJ and ΨHJ breakpoint signatures of the episomal reintegration are depicted.</p> <p>(C) V(D)J-mediated translocation generating one <i>trans</i>-chromosomal CJ and one <i>trans</i>-CSJ.</p> <p>(D) Ongoing V(D)J recombination of a CSJ in <i>cis.</i> The illustration depicts a CSJ generated through V(D)J-mediated translocation, but the same principle stands for a CSJ generated through V(D)J recombination by inversion.</p> <p>White triangles, 12-RSS; black triangles, 23-RSS; dotted white triangles, IG/TCR or cryptic 12-RSS; rectangles, IG/TCR gene segment or oncogene activated through rearrangement; ellipses, RAG-1/2. Indicative chromosomes and derivative chromosomes are shown. Dented line in the ΨHJ and CJ represents processing at the junction (Δ, nucleotide deletion; P, P nucleotide addition; N, N nucleotide addition).</p></div

Ex Vivo <i>trans</i>-V(D)J Recombination Assay

<div><p>(A) Schematic representation of <i>trans</i>-V(D)J recombination between an ESJ (donor) and a 12-RSS (target). The expected breakpoints (one SJ and one ΨHJ) are shown. The various donor/target combinations assayed are boxed. PCR primers are depicted by arrows. Nested IR800-labeled PE primers are indicated by an asterisk.</p> <p>(B) Typical example of PCR/PE assays obtained from the (Dβ1Δ) ESJ × Jβ2.7 12-RSS combination. The expected sizes of the PE products are indicated. As the resolution of the PE assay is to the base, ΨHJ patterns typically show multiple bands (corresponding to the spectra of nucleotide addition and deletion), while SJ patterns are typically centered on a major band (corresponding to no or limited nucleotide processing). PE assays shown were performed on 0.75 μl PCR. Five independent PCRs from two independent transfections performed with (T3–T4) or without RAG-1/2 (T1–T2) are shown for each junction.</p></div

Breakpoint Sequences of Ex Vivo <i>trans</i>-V(D)J Recombinants

<p>SJ (primer combination 3 + 2). Top and bottom lanes depict sequences before recombination (heptamers, spacers and nonamers are specified). Recombined clones are depicted between top and bottom lanes, with homology to the unrecombined sequences indicated by vertical lines. Lower cases represent reactive RSS involved in the <i>trans</i>-V(D)J recombination reactions (specified as donor RSS and target RSS). Upper cases represent coding segments, or bystander RSSs in the ESJ behaving like coding segments (specified as bystander RSSs). Italics indicate potential P nucleotides; bold type, N nucleotides; heptamers and nonamers are underlined; identical sequences shown on separate lanes are issued from distinct transfections and represent therefore independent junctions. Since identical sequences were often obtained in a given transfection, this representation might in some cases bias the representation towards processed junctions (because they acquire distinguishing features).</p

Semiquantitative Ex Vivo <i>trans</i>-V(D)J Recombination Assay

<div><p>(A) Schematic representation of the comparative <i>trans</i>-V(D)J recombination assay. Left panel: recombination of an ESJ 23-RSS (Jδ1/Dδ3 or Dβ1Δ) with a 12-RSS target (Jβ2.7); Right panel: recombination of a coding segment/23-RSS (Dδ3 or Dβ1) with a 12-RSS target (Jβ2.7). The expected breakpoints are shown for each case.</p> <p>(B) Typical example of semiquantitative PCR/PE assays obtained from the (Jδ1/Dδ3) ESJ × Jβ2.7 versus Dδ3 × Jβ2.7 comparisons. PE assays shown were performed on serial dilutions of PCR products.</p></div

Strategy for In Vivo Detection of Episomal Reintegration in the (Dβ1^GFP × Eβ⁻) double-mutant (DE) mouse

<div><p>(A) TCRβ allelic configuration of the DE mouse. The Eβ knockout prevents efficient rearrangements on allele 1, and the GFP knockin in Dβ1 prevents D-J cluster 1 rearrangements on allele 2. In this configuration, all productive TCRβ chains are issued from Vβ rearrangements to D-J cluster 2 of allele 2. Consequently, TCRβ chain production and translocation to the TCRβ locus are mutually exclusive. The generation of a TCR excision circle carrying a (Jβ2/Dβ2)ESJ through Dβ2-Jβ2 rearrangements from the Dβ1^GFP allele is depicted.</p> <p>(B) Reintegration of (Jβ2/Dβ2) ESJs in Jδ1. In TCRαβ⁺ cell-sorted thymocytes from DE mice, the presence of a Jβ2.7/Jδ1 ΨHJ specifically signs the reintegration of (Jβ2/Dβ2)ESJ in Jδ1, and is assayed by fluctuation PCR.</p></div