Fig. 2.

Evaluation of the number of clonotypic B cells with the rearranged VDJ locus in the xenograft. a A scheme of VDJ locus differentiation during recombination. b PCR. Tumor—PCR with three sets of degenerate primers (JH in each case, and FR2b, FR3c, and FR1c) for conservative regions of the VDJ locus with DNA isolated from the tumor graft. Liver—DNA isolated from the liver of mice; the presence of the fragment indicates the presence of many identical B clones in the DNA sample. c Sequencing of the rearranged B clone. Comparison of the sequences of several sequenced clones (figure shows 9 out of 23). The V, D, and J segments are shown in colors corresponding to those in Scheme A. The arrow indicates the primer sequence. The brackets indicate the site for cloning into the plasmid PUC19 (at the EcoRV site). The sequences are identical. Individual substitutions are shown in yellow. d qPCR with DNA isolated from the tumor graft. The plot shows calibration curves for human DNA (Rplp0 primers, total human DNA as a template) and mouse DNA (PTGER2 primers, DNA from mouse bone marrow cells as a template). Dots on the calibration curves indicate the amount of different DNAs in the tumor graft. Human DNA—67.6 ng, mouse DNA—22.0 ng. e qPCR quantification of B clone copies in DNA isolated from the tumor graft. The calibration curve was obtained by titration of B clone DNA (500 bp) and JH/FR3c primers. The dot denotes the number of B clones in studied DNA, which is calculated using the calibration curve slope