Creation of mice bearing a partial duplication of HPRT gene marked with a GFP gene and detection of revertant cells in situ as GFP-positive somatic cells

It is becoming clear that apparently normal somatic cells accumulate mutations. Such accumulations or propagations of mutant cells are thought to be related to certain diseases such as cancer. To better understand the nature of somatic mutations, we developed a mouse model that enables in vivo detection of rare genetically altered cells via GFP positive cells. The mouse model carries a partial duplication of 3' portion of X-chromosomal HPRT gene and a GFP gene at the end of the last exon. In addition, although HPRT gene expression was thought ubiquitous, the expression level was found insufficient in vivo to make the revertant cells detectable by GFP positivity. To overcome the problem, we replaced the natural HPRT-gene promoter with a CAG promoter. In such animals, termed HPRT-dup-GFP mouse, losing one duplicated segment by crossover between the two sister chromatids or within a single molecule of DNA reactivates gene function, producing hybrid HPRT-GFP proteins which, in turn, cause the revertant cells to be detected as GFP-positive cells in various tissues. Frequencies of green mutant cells were measured using fixed and frozen sections (liver and pancreas), fixed whole mount (small intestine), or by means of flow cytometry (unfixed splenocytes). The results showed that the frequencies varied extensively among individuals as well as among tissues. X-ray exposure (3 Gy) increased the frequency moderately (~2 times) in the liver and small intestine. Further, in two animals out of 278 examined, some solid tissues showed too many GFP-positive cells to score (termed extreme jackpot mutation). Present results illustrated a complex nature of somatic mutations occurring in vivo. While the HPRT-dup-GFP mouse may have a potential for detecting tissue-specific environmental mutagens, large inter-individual variations of mutant cell frequency cause the results unstable and hence have to be reduced. This future challenge will likely involve lowering the background mutation frequency, thus reducing inter-individual variation

Fetal irradiation of rats induces persistent translocations in mammary epithelial cells similar to the level after adult irradiation, but not in hematolymphoid cells.

In both humans and mice, fetal exposure to radiation fails to induce a persistent increase in the frequency of chromosome aberrations in blood lymphocytes. Such a low-level response to radiation exposure is counterintuitive in view of the generally accepted belief that a fetus is sensitive to radiation. To determine if this is a general phenomenon, both mammary epithelial cells and spleen cells were studied in rats. Fetuses of 17.5 days postcoitus were irradiated with 2 Gy of gamma rays, and mammary tissues were removed 6-45 weeks later. Subsequently, short-term cultures were established to detect translocations using the two-color FISH method. The results showed that translocation frequencies were not only elevated in rats irradiated as fetuses, but were also almost as high as those in rats that were irradiated as adults (12 weeks old, pregnant mothers or young virgins) and examined 6-45 weeks later. There was no evidence of higher sensitivity in fetal cells with respect to the induction of translocations. In contrast, translocation frequencies in spleen cells were not elevated in adult rats irradiated as fetuses but were increased after irradiation of adults as previously seen in mouse spleen cells and human T lymphocytes. In the case of irradiation of adult rats, the induced translocation frequencies were similar between spleen cells and mammary epithelial cells. If we take translocation frequency as a surrogate marker of potential carcinogenic effect of radiation, the current results suggest that fetal irradiation can induce persistent potential carcinogenic damage in mammary stem/progenitor cells but this does not contribute to the increased risk of cancer since it has been reported that irradiation of fetal rats of the SD strain does not increase the risk of mammary cancers. Possible reasons for this discrepancy are discussed

RAD18 activates the G2/M checkpoint through DNA damage signaling to maintain genome integrity after ionizing radiation exposure.

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure

Tissues with extreme jackpot mutations.

<p><b>(A)</b> A low magnification view of small intestine under a dissecting fluorescent microscopy. Many mutant green villi are seen. <b>(B)</b> A cross section of intestinal villi. Note that mutant GFP-positive cell clusters (bright green sectors) are present in most of the villi. <b>(C)</b> A cross section of liver. (<b>D</b>) An enlarged picture of (<b>C</b>), bar = 100 μm in both (<b>C)</b> and (<b>D</b>).</p

Distributions of mutational events per 10⁶ cells examined.

<p>In the liver <b>(A)</b> and pancreas <b>(B)</b>, frozen sections were scored under a fluorescent microscopy and a cluster of GFP-positive cells that were interrelated across slices were counted as a single event. In small intestine <b>(C)</b>, green streaks that appeared in villi were scored using a dissecting microscopy. The results were used to estimate mutational events per 10⁶ crypt stem cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136041#sec002" target="_blank">Materials and Methods</a>). In spleen (<b>D</b>), mutant cells were detected by a flow cytometer and the frequency was expressed per 10⁶ cells. Open and filled circles represent animals receiving 0-Gy and 3-Gy irradiation, respectively. Triangles and squares represent means and medians, respectively. Asterisks (panel D) represent extreme outliers, with their actual values provided in the figure. No extreme jackpot mutations were detected in this series of the experiments.</p

Mean mutation frequency in 10⁻⁶ compared between control (0 Gy) and irradiated (3 Gy) mice.

<p>*Wilcoxon <i>z</i>-values: intestine, 3.35; liver, 2.43; pancreas, 0.45; spleen, 1.11.</p><p>Means were compared with a Wilcoxon-Mann-Whitney test. Fold increase (irradiated vs. control), along with bootstrapped 95% CIs, are also presented; CIs not containing 1 indicate statistical significance at α = 0.05.</p

Outline of the HPRT-dup-GFP system and in vivo detection of revertant cells.

<p><b>A)</b> Genetic composition of theHPRT-dup-GFP system, <b>B-D</b>) Frozen sections of liver, <b>E-H</b>) frozen sections of pancreas. Note that nuclei of background GFP-negative cells exhibit yellowish color derived from auto-fluorescence. <b>F</b>) and <b>H</b>) are enlarged views of <b>E</b>) and <b>G</b>), respectively (scale bar = 100 μm). <b>I-M)</b> whole mount of small intestine; specifically, <b>J</b> represent an isolated villus with single mutant streaks (bar = 100 μm), <b>K)</b> an example of a whole villus composed of green mutant cells, <b>L) and M</b>) are villi of small intestinal with mutant streaks in two adjacent villi (bar = 100 μm). (Note that E and F are squashed samples of pancreas. In these, network of capillary blood vessels is visible.) Arrows indicate mutant(s).</p