In order to maximize the mutagenic effectiveness of N-ethyl-N-nitrosourea in mouse stem-cell spermatogonia, advantage was taken of the fact that these cells can accumulate mutations from repeated doses given over relatively long time periods. Repeated doses (100 mg/kg) of ethylnitrosourea injected intraperitoneally into male mice at weekly intervals were found to allow adequate survival and fertility with total dosages of 300 and 400 mg/kg. The specificlocus mutation frequencies at these dosages were, respectively, 1.8 and 2.2 times that obtained with the maximal practicable single dose of 250 mg/kg. The mutation frequency induced by a 400 mg/kg dosage of ethylnitrosourea is 12 times the maximal mutation frequency achievable with a single exposure to x-rays and 36 times that reported for procarbazine, the most effective chemical mutagen previously known for mouse stem-cell spermatogonia. Ethylnitrosourea is already the mutagen of choice in deliberate attempts to create mouse models for human disease and in any experiments in which a maximal mutation rate is desired. Repeated-dose regimens similar to the ones reported here should increase the efficiency of such studies.One advantage of determining induced mutation rates in stem-cell spermatogonia is that these cells can accumulate mutational lesions throughout the reproductive life span of the animal; therefore, repeated doses of mutagens can be given over relatively long time periods to amass a total dosage higher than that tolerated in a single exposure. Ethylnitrosourea (EtNU) has been shown to be the most effective mutagen for induction of specific-locus mutations in mouse spermatogonia (1,2). This has led rapidly to its being the mutagen of choice by many investigators (3,4). Therefore, it seemed useful to test whether repeated doses could be given to yield higher specific-locus mutation rates than those obtained by single exposures. This paper presents data from specific-locus tests with repeated doses of EtNU and from survival and fertility tests that were conducted prior to the specific-locus tests in order to choose feasible dosage regimens. A brief abstract of the preliminary results on survival and fertility has appeared (5). MATERIALS AND METHODSSurvival and Fertility Tests. EtNU, synthesized in our laboratory by D. G. Doherty, was dissolved in phosphate buffer (6) adjusted to pH 6. The dose injected intraperitoneally was matched to the body weight of the animals by adjusting the volume of solution injected, which never exceeded 1 ml. Male (101 x C3H)F1 mice were injected with dosages of 300 or 400 mg of EtNU/kg, given in 100 mg/kg doses. In four separate experiments, the individual doses were spaced 1 day, 2 days, 4 days, or 7 days apart, respectively. Six mice, 12 weeks old at the first injection, were in each dosage group in each experiment. All injections were completed within 1 hr after the EtNU was dissolved. Seven weeks after the final injection, these males were mated to females of our standard specific-locus test strain, T, which is h...

The mammalian Nell1 gene encodes a protein kinase C-beta1 (PKC-beta1) binding protein that belongs to a new class of cell-signaling molecules controlling cell growth and differentiation. Over-expression of Nell1 in the developing cranial sutures in both human and mouse induces craniosynostosis, the premature fusion of the growing cranial bone fronts. Here, we report the generation, positional cloning and characterization of Nell1(6R), a recessive, neonatal-lethal point mutation in the mouse Nell1 gene, induced by N-ethyl-N-nitrosourea. Nell1(6R) has a T-->A base change that converts a codon for cysteine into a premature stop codon [Cys(502)Ter], resulting in severe truncation of the predicted protein product and marked reduction in steady-state levels of the transcript. In addition to the expected alteration of cranial morphology, Nell1(6R) mutants manifest skeletal defects in the vertebral column and ribcage, revealing a hitherto undefined role for Nell1 in signal transduction in endochondral ossification. Real-time quantitative reverse transcription-PCR assays of 219 genes showed an association between the loss of Nell1 function and reduced expression of genes for extracellular matrix (ECM) proteins critical for chondrogenesis and osteogenesis. Several affected genes are involved in the human cartilage disorder Ehlers-Danlos Syndrome and other disorders associated with spinal curvature anomalies. Nell1(6R) mutant mice are a new tool for elucidating basic mechanisms in osteoblast and chrondrocyte differentiation in the developing skull and vertebral column and understanding how perturbations in the production of ECM proteins can lead to anomalies in these structures.

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.

Hereditary tyrosinemia type 1 (HT1) is a severe autosomal recessive metabolic disease associated with point mutations in the human fumarylacetoacetate hydrolase (FAH) gene that disrupt tyrosine catabolism. An acute form of HT1 results in death during the first months of life because of hepatic failure, whereas a chronic form leads to gradual development of liver disease often accompanied by renal dysfunction, childhood rickets, neurological crisis, and hepatocellular carcinoma. Mice homozygous for certain chromosome 7 deletions of the albino Tyr; c locus that also include Fah die perinatally as a result of liver dysfunction and exhibit a complex syndrome characterized by structural abnormalities and alterations in gene expression in the liver and kidney. Here we report that two independent, postnatally lethal mutations induced by N-ethyl-N-nitrosourea and mapped near Tyr are alleles of Fah. The Fah 6287SB allele is a missense mutation in exon 6, and Fah 5961SB is a splice mutation causing loss of exon 7, a subsequent frameshift in the resulting mRNA, and a severe reduction of Fah mRNA levels. Increased levels of the diagnostic metabolite succinylacetone in the urine of the Fah 6287SB and Fah 5961SB mutants indicate that these mutations cause a decrease in Fah enzymatic activity. Thus, the neonatal phenotype present in both mutants is due to a deficiency in Fah caused by a point mutation, and we propose Fah 5961SB and Fah 6287SB as mouse models for acute and chronic forms of human HT1, respectively.