Dedicated high-resolution small animal imaging systems have recently emerged as important new tools for cancer research. These new imaging systems permit researchers to noninvasively screen animals for mutations or pathologies and to monitor disease progression and response to therapy. One imaging modality, X-ray microcomputed tomography (microCT) shows promise as a cost-effective means for detecting and characterizing soft-tissue structures, skeletal abnormalities, and tumors in live animals. MicroCT systems provide high-resolution images (typically 50 microns or less), rapid data acquisition (typically 5 to 30 minutes), excellent sensitivity to skeletal tissue and good sensitivity to soft tissue, particularly when contrast-enhancing media are employed. The development of microCT technology for small animal imaging is reviewed, and key considerations for designing small animal microCT imaging protocols are summarized. Recent studies on mouse prostate, lung and bone tumor models are overviewed.

Use of the specific-locus test to measure the frequency of transmitted gene mutations induced in mouse spermatogonia has shown ethylnitrosourea to be by far the most potent mutagen yet discovered in the mouse. The dose used, 250 mg/kg, gave a mutation rate 5 times as high as had been obtained with 600 R, the most effective acute dose of x-rays. Compared to procarbazine, heretofore the most mutagenic chemical known in the mouse, ethylnitrosourea proved to be 15 times more mutagenic than the peak effect obtained with the most effective dose of procarbazine. Because of its high mutagenicity, ethylnitrosourea can serve as a model compound in exploring the effect of such factors as dose response, dose fractionation, sex, and cell stage on the mutagenic action of a chemical. Ethylnitrosourea is clearly the mutagen of choice for the production of any kind of desired new gene mutations in the mouse. More than a score of chemicals, most of them well-known mutagens in other organisms, have been tested by the mouse specific-locus method. Only three, however, have shown a clear-cut positive mutagenic effect in treated mouse spermatogonial stages (1), and the most effective dose of the most potent of these three-namely, procarbazine (2)-produced only approximately one-third as many mutations as had been obtained with 600 R (1 R = 2.6 X 10-4 coulombs/kg) of acute x-irradiation. The impression was developing that perhaps the mammalian testis was efficiently protected against damage by chemicals. The possibility also existed that the spermatogonia might be effective at repairing mutational damage. It began to appear possible that perhaps no chemical could break through the mammalian body's defense barriers to produce more than a moderate mutagenic effect in spermatogonia.Although repair and other defense mechanisms may still prove to operate against many substances, the present results show that N-ethyl-N-nitrosourea (ENU) provides a dramatic exception.MATERIALS AND METHODS ENU (purchased from Bio-Clinical Laboratories, Bohemia, NY) was dissolved in phosphate buffer (3) adjusted to pH 6. The dose injected intraperitoneally was matched to the body weight of each animal by adjusting the volume of the injected solution, which never exceeded 1 ml. Wild-type (101XC3H)Fl male mice 9-19 weeks old were injected with a single dose (250 mg/kg) of ENU. All animals given this dose survive, whereas a 300-mg/kg dose kills about 40%. All injections were completed within less than 1 hr after the chemical was dissolved. The injected males were mated to females of our standard specific-locus test strain (T) which is homozygous for seven marker genes (4). Each male was mated to a group of either two or four females and moved to a new group of females each week. After each 7-week period the males were rotated back to the original group of females to start the cycle over again. The offspring were scored for mutations at the seven loci. RESULTS AND DISCUSSIONThe 250-mg/kg dose induced a long period of sterility. The males were fertile for a...

Genetic reference populations in model organisms are critical resources for systems genetic analysis of disease related phenotypes. The breeding history of these inbred panels may influence detectable allelic and phenotypic diversity. The existing panel of common inbred strains reflects historical selection biases, and existing recombinant inbred panels have low allelic diversity. All such populations may be subject to consequences of inbreeding depression. The Collaborative Cross (CC) is a mouse reference population with high allelic diversity that is being constructed using a randomized breeding design that systematically outcrosses eight founder strains, followed by inbreeding to obtain new recombinant inbred strains. Five of the eight founders are common laboratory strains, and three are wild-derived. Since its inception, the partially inbred CC has been characterized for physiological, morphological, and behavioral traits. The construction of this population provided a unique opportunity to observe phenotypic variation as new allelic combinations arose through intercrossing and inbreeding to create new stable genetic combinations. Processes including inbreeding depression and its impact on allelic and phenotypic diversity were assessed. Phenotypic variation in the CC breeding population exceeds that of existing mouse genetic reference populations due to both high founder genetic diversity and novel epistatic combinations. However, some focal evidence of allele purging was detected including a suggestive QTL for litter size in a location of changing allele frequency. Despite these inescapable pressures, high diversity and precision for genetic mapping remain. These results demonstrate the potential of the CC population once completed and highlight implications for development of related populations.

X-linked dominant disorders that are exclusively lethal prenatally in hemizygous males have been described in human and mouse. None of the genes responsible has been isolated in either species. The bare patches (Bpa) and striated (Str) mouse mutations were originally identified in female offspring of X-irradiated males. Subsequently, additional independent alleles were described. We have previously mapped these X-linked dominant, male-lethal mutations to an overlapping region of 600 kb that is homologous to human Xq28 (ref. 4) and identified several candidate genes in this interval. Here we report mutations in one of these genes, Nsdhl, encoding an NAD(P)H steroid dehydrogenase-like protein, in two independent Bpa and three independent Str alleles. Quantitative analysis of sterols from tissues of affected Bpa mice support a role for Nsdhl in cholesterol biosynthesis. Our results demonstrate that Bpa and Str are allelic mutations and identify the first mammalian locus associated with an X-linked dominant, male-lethal phenotype. They also expand the spectrum of phenotypes associated with abnormalities of cholesterol metabolism.

The germline supermutagen, N-ethyl-N-nitrosourea (ENU), has a variety of effects on mice. ENU is a toxin and carcinogen as well as a mutagen, and strains differ in their susceptibility to its effects. Therefore, it is necessary to determine an appropriate mutagenic, non-toxic dose of ENU for strains that are to be used in experiments. In order to provide some guidance, we have compiled data from a number of laboratories that have exposed male mice from inbred and non-inbred strains or their F(1) hybrids to ENU. The results show that most F(1) hybrid animals tolerate ENU well, but that inbred strains of mice vary in their longevity and in their ability to recover fertility after treatment with ENU.

The mammalian testis expresses a class of small noncoding RNAs that interact with mammalian PIWI proteins. In mice, the PIWI-interacting RNAs (piRNAs) partner with mammalian PIWI proteins, PIWIL1 and PIWIL2, also known as MIWI and MILI, to maintain transposon silencing in the germline genome. Here, we demonstrate that inactivation of Nct1/2, two noncoding RNAs encoding piRNAs, leads to derepression of LINE-1 (L1) but does not affect mouse viability, spermatogenesis, testicular gene expression, or fertility. These findings indicate that piRNAs from a cluster on chromosome 2 are necessary to maintain transposon silencing.

The chemotherapeutic agent chlorambucil was found to be more effective than x-rays or any chemical investigated to date in inducing high yields of mouse germ-line mutations that appear to be deletions or other structural changes. Induction of mutations involving seven specific loci was studied after exposures of various male germ-cell stages to chlorambucil at 10-25 mg/kg. A total of 60,750 offspring was scored. Mutation rates in spermatogonial stem cells were not significantly increased over control values; this negative result is not attributable to selective elimination of mutant cells. Mutations were, however, clearly induced in treated poststem-cell stages, among which marked variations in mutational response were found. Maximum yield occurred after exposure of early spermatids, with -1% of all offspring carrying a specific-locus mutation in the 10 mg/kg group. The stageresponse pattern for chlorambucil differs from that of all other chemicals investigated to date in the specific-locus test. Thus far, all but one of the tested mutations induced by chlorambucil in post-stem-cell stages have been proved deletions or other structural changes by genetic, cytogenetic, and/or molecular criteria. Deletion mutations have recently been useful for molecular mapping and for structure-function correlations of genomic regions. For generating presumed large-lesion germline mutations at highest frequencies, chlorambucil may be the mutagen of choice.Chromosomal deletions have been valuable in the molecular mapping of genetic loci in humans (1, 2). In the mouse, the existence of large arrays of radiation-induced deletion mutations involving marked loci has been an important factor in recent advances in structurally and functionally characterizing regions of the genome that surround these loci (for review, see ref.3). Multilocus deletions constitute a relatively high proportion of mutations induced by certain radiation treatments (4). Although the total yield of mutations produced by N-ethyl-N-nitrosourea (EtNU) considerably exceeds the yield of radiation-induced mutations (5), EtNU induces chiefly intragenic lesions, including base-pair changes (4, 6). This paper reports the finding that a chemical mutagen, chlorambucil, is considerably more effective than radiation or any chemical investigated to date in inducing high yields of germ-line mutations that appear to be multilocus deletions or other structural changes.Chlorambucil (7). Chlorambucil is a germ-cell mutagen in Drosophila, where it induces very high frequencies of sex-linked recessive lethals as well as translocations in meiotic and postmeiotic stages (8). Evidence that chlorambucil reaches the testis of mice comes from the results of histological studies (9), in which exposure to this chemical at 20 mg/kg was moderately to strongly cytotoxic to differentiating spermatogonia, and slightly toxic to spermatogonial stem cells. In this study, mutagenicity of chlorambucil was tested in male germ-cell stages of the mouse, ranging from spermatogonial stem cells t...

The extreme mutagenic effectiveness ofN-ethyl-N-nitrosourea in the mouse has permitted the accumulation of the most extensive dose-response data yet obtained for chemical induction of specific-locus mutations in spermatogonia. In the lower portion of the curve, below a dose of 100 mg/kg, the data fall statistically significantly below a maximum likelihood fit to a straight line. Independent evidence indicates that, over this dose range, ethylnitrosourea reaches the testis in amounts directly proportional to the injected dose. It is concluded that, despite the mutagenic effectiveness of ethylnitrosourea, the spermatogonia are apparently capable of repairing at least a major part of the mutational damage when the repair process is not swamped by a high dose. This finding is important both in basic studies on the mutagenic action of chemicals in mammals and in risk estimation.In 1979, it was shown (1) with the specific-locus method that N-ethyl-N-nitrosourea (ENU) is, by more than an order ofmagnitude, a more effective mutagen in mouse spermatogonia than any other compound tested. It was pointed out that ENU could, therefore, serve as a model chemical in exploring, with relative ease, the various factors-such as dose, dose fractionation, sex, and cell stage-that might affect mutagenic action. Preliminary findings on the effects of these and other factors have been reported (2-7). A more detailed dose-response curve than that obtained for any other compound tested for transmitted mutations in the mouse is now available and is presented here.MATERIALS AND METHODS ENU (purchased from Bio-Clinical Laboratories, Bohemia, NY) was dissolved in phosphate buffer (8) adjusted to pH 6. The dose injected intraperitoneally was matched to the body weight of the animal by adjusting the volume of solution injected, which approximated 1 ml for all doses. Wild-type (101 x C3H)F1 male mice 11.5-14.5 wk old were injected with a single dose. The experiment was done in two series, the first with doses of 100, 150, 200, and 250 mg/kg, and the second, 11 wk later, with doses of 25, 50, 75, and 100 mg/kg. The same crystalline batch of ENU, kept in a dessicator under refrigeration, was used throughout but, to check for deterioration of the chemical or other change in the conditions, the 100-mg/kg dose was included in both series. All injections were completed within 2 hr after the chemical was dissolved. The injected males were mated to females of our standard specific-locus test strain (T), which is homozygous for seven marker genes (9). Each male was mated to a group of either two or four females and moved to a new group of females each week. After each 7-wk period, the males were rotated back to the original group offemales to start the cycle over again. All the offspring reported here came from conceptions occurring more than 7 wk after injection, thus ensuring that they were derived from cells that had been exposed to the chemical in spermatogonial stem-cell stage. The offspring were scored for mutations at the seven loci. RES...