Spi? selection: An efficient method to detect ?-ray-induced deletions in transgenic mice

Abstract

Despite the importance of genome rearrangement in the etiology of cancer and human genetic disease, deletion mutations are poorly detectable by transgenic rodent mutagenicity tests. To facilitate the detection and molecular analysis of deletion mutations in vivo, we established a transgenic mouse model harboring a λEG10 shuttle vector that includes the red and gam genes for Spi− (sensitive to P2 interference) selection [Nohmi et al. (1996] Environ. Mol. Mutagen. 28:465–470]. This selection has a great advantage over other genetic systems, because phage deletion mutants can be preferentially selected as Spi− plaques, which can then be subjected to molecular analysis. Here, we show nucleotide sequences of 41 junctions of deletion mutations induced by γ‐irradiation. Unlike spontaneous deletion mutants, more than half of the large deletions occurred between short homologous sequences from one to eight bp. The remaining junctions had no such homologous sequences. Intriguingly, two Spi− mutants had P (palindrome)‐like nucleotide additions at the breakpoints, which are frequently observed in the coding junctions of V(D)J recombination, suggesting that broken DNA molecules with hairpin structures can be intermediates in the repair of radiation‐induced double‐strand breaks. We conclude that Spi− selection is useful for the efficient detection of deletion mutations in vivo and that most rearrangements induced by γ‐rays in mice are mediated by illegitimate recombination through DNA end‐joining. Environ. Mol. Mutagen. 34:9–15, 1999 © 1999 Wiley‐Liss, Inc.

A Single Neonatal Exposure to Aflatoxin B1 Induces Prolonged Genetic Damage in Two Loci of Mouse Liver

“…The sequence characteristics of deletions found in gpt delta mice after treatment with various agents include -1 bp frameshifts and larger deletions (> 10 kb) containing junction sequences of limited homology that could result from end joining of double strand breaks (Horiguchi et al, 2001;Masumura et al, 2000;Nohmi et al, 1999). The molecular characteristics of the Spi -mutants from the liver of AFB 1 -treated mice differ from those found in the aforementioned studies.…”

Spontaneous Mutagenesis in Rodents: Spontaneous Gene Mutations Identified by Neutral Reporter Genes in gpt Delta Transgenic Mice and Rats

“…The gpt and Spi − assay systems have been validated primarily in mice with many chemical mutagens/carcinogens, UV and ionizing radiation, for which mutagenicity, organ specificity and mutation spectrum have been thoroughly characterized. 1,[10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] Recently, the outbred gpt delta SD rat was backcrossed with Fisher 344 (F344) rat, to establish an inbred gpt delta rat (F344). In this review, we focus on the spontaneous mutations detected by the gpt and Spi − assays in gpt delta mice and rats and discuss possible mechanisms underlying these in vivo mutations.…”

“…However, translation of the gam and red genes is probably linked, and the gam gene is first transcribed so that the 1 bp deletions in the gam gene may interfere with the start of translation of the downstream red gene, thereby functionally inactivating not only gam but also red. 13) The percentages of the 1 bp deletions are from 50-77%. But these values may be underestimated because we regard identical mutations recovered from the same tissue samples to have resulted from clonal expansion and count them as a single mutation.…”

Alteration of Genome Structure Induced by Very Low Dose-Rate Irradiation in Mouse Tissues

“…The gpt-delta mice containing lambda EG10 genomic DNA (Nohmi, Suzuki, Masumura, Yamada, Matsui, Ueda, et al 1999) were mated to SWR mice (Shaver-Walker, Urlando, Tao, Zhang, & Heddle, 1995) to obtain F1 mice, who contained both the red·gam gene and Dlb1 gene, which can be analysed separately. A schematic illustration of a gpt-delta mouse is shown in Figure 1.…”