Unniraman et al. reply:

The generation of Igh switch region–Myc (Igh-Myc) translocations is thought to be a primary step in the development of mouse plasmacytomas1,2. Ramiro et al. recently reported the detection of Igh-Myc translocations in wild-type but not AID-deficient (Aicda−/−) Il6-transgenic mice using a long template PCR assay with primers specific for Myc and Igh Cμ (ref. 3). In contrast, we detected equal frequencies of translocations in unmanipulated wild-type and Aicda−/− mice4 using a consensus primer PCR assay that takes advantage of primers that hybridize to multiple locations in Sμ and Sα (ref. 5).

The consensus primer PCR assay is more sensitive2,5,6, whereas the long template PCR assay allows detection of the two reciprocal translocation products in mice in which translocation-bearing cell populations have been triggered to expand2,7,8,9. Before expansion, cells containing these translocations are rare, making them difficult to detect in duplicate assays of the same DNA sample4,5,6. Translocations are also detected in strains of mice that are resistant to plasmacytomagenesis, suggesting that resistance in these strains arises at a later step in the disease process, probably during expansion of translocation-bearing cell populations5,6. Although translocations have been detected in unmanipulated wild-type mice using both the long template and consensus primer PCR assays, they are more readily detected using the latter assay2,5,6,10,11. Ramiro et al. now report that Igh-Myc junction products can be amplified from wild-type kidney and the gut of Rag1−/− mice with the consensus primer PCR assay. These data contrast with our finding that no products could be obtained from brain or kidney with this assay4. We have now evaluated 30 gut and 5 spleen DNA samples obtained from five C57BL/6 Rag1−/− mice using the consensus primer PCR assay (Fig. 1). None of these 35 samples, assayed in duplicate, yielded a positive signal in any reaction (Fig. 1a and c, lanes 1–16, and d, lanes 1–3, and data not shown). In addition, 25 BALB/c brain and kidney samples, assayed once in parallel, were uniformly negative (Fig. 1b, lanes 5–16, and data not shown). In the same set of reactions, DNA samples from untreated mice (Fig. 1b, lanes 1–4, d, lanes 5–8, and data not shown) that had a positive score in previous assays yielded a positive signal at the expected frequency (6 positive results in 15 reactions; Fig. 1b, lane 4, and d, lane 7, and data not shown), including four samples from untreated Aicda−/− mice (Fig. 1b, lane 4, and data not shown).

Figure 1: PCR and Southern blot assays of samples from Rag1−/− mice as well as positive and negative controls.
figure 1

Reactions were done as reported4. All reactions were done at the same time and data represent parts of the same gels that were split only for the convenience of labeling. Data are representative reactions with DNA from 30 gut and 5 spleen (S) samples from five Rag1−/− mice (a, lanes 1–16, c, lanes 1–16 and d, lanes 1–3). Data also include representative reactions done in parallel with eight kidney and four brain samples from Aicda+/+, Aicda−/− and BALB/c mice (b, lanes 5–16) and six gut and two Peyer's patch samples from unmanipulated Aicda+/+, Aicda+/− and Aicda−/− mice (b, lanes 1–4 and d5-8), as well as eight representative products (from three Rag1−/− samples and three previously positive and two brain and kidney samples) after the first 40 cycles of PCR (d, lanes 9–16). NT, no template. Membranes were exposed to a phosphorimager screen for 16 h.

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Although we do not have a ready explanation for the discrepancy between our results and those of Ramiro et al., we note that the consensus primer PCR assay is very sensitive and that variations in reaction conditions might result in the generation of artifacts. The only previously reported artifact arising from consensus primer PCR assays was the amplification of switch-switch products5, but our assay does not detect such products because of our inclusion of a hybridization step with a Myc-specific probe. We did detect faint smears in some samples that were less than 2% as strong as the positive signals (for example, Fig. 1b, lanes 2, 10 and16). However, we have never succeeded in cloning any Igh-Myc junctions from such reactions and therefore have consistently assigned them negative scores. Our inability to detect positive signals from many brain and kidney samples or from tissues from Rag1−/− mice, together with our published data4, provide molecular evidence for the existence of Igh-Myc translocations in wild-type and Aicda−/− mice. It will now be necessary to develop alternative types of assays capable of detecting rare translocation-positive cells.