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Aim of this review is to demonstrate the relevance of animal models created by ENU mutagenesis for the pharmaceutical community to understand diseases and the modulation of disease status by pharmaceutical compounds. We give an overview of what ENU mutagenesis in mice implies and introduce the main research centers running ENU mutagenesis projects. The different strategies of ENU mutagenesis screens are explained as well as the latest advances in mapping and mutation detection strategies, which until recently have been the main limiting step in forward genetics/ phenotype- driven approaches. ENU mutagenesis in mice has shown its power by providing animal models for human monogenic diseases. Moreover, the development of modifier and sensitized screens extended this resource to models for multigenic diseases and thereby opened the perspective to understand the modulation of disease states. Finally, we provide information about the accessibility and availability of these models for academic research.

The aim of this study was the application of a phenotype-driven N-ethyl-N-nitrosourea (ENU) mutagenesis screen in mice for the identification of dominant mutations involved in the regulation and modulation of alcohol-drinking behavior. The chemical mutagen ENU was utilized in the generation of 131 male ENU-mutant C57BL/6J mice (G0). These ENU-treated mice were paired with wild-type C57BL/6J mice to generate G1 and subsequent generations. In total, 3327 mice were generated. Starting with G1, mice were screened for voluntary oral self-administration of 10% (v/v) alcohol vs. water in a two-bottle paradigm. From these mice, after a total period of 5 weeks of drinking, 43 mutants fulfilled the criteria of an "alcohol phenotype," that is, high or low ethanol intake. They were then selected for breeding and tested in a "confirmation cross" (G2-G4) for inheritance. Although we did not establish stable high or low drinking lines, several results were obtained in the context of alcohol consumption. First, female mice drank more alcohol than their male counterparts. Second, the former demonstrated greater infertility. Third, all animals displayed relatively stable alcohol intake, although significantly different in two different laboratories. Finally, seasonal and monthly variability was observed, with the highest alcohol consumption occurring in spring and the lowest in autumn. In conclusion, it seems difficult to identify dominant mutations involved in the modulation or regulation of voluntary alcohol consumption via a phenotype-driven ENU mutagenesis screen. In accordance with the findings from knockout studies, we suggest that mainly recessive mutations contribute to an alcohol-drinking or alcohol-avoiding phenotype.

A novel ENU-induced mutation in the mouse leading to a nuclear and zonular opacity of the eye lens (Aey1) was mapped to chromosome 1 between the markers D1Mit303 and D1Mit332. On the basis of the chromosomal position, the γ-crystallin encoding gene cluster (Cryg) and the βA2-crystallin encoding gene Cryba2 were tested as candidate genes. An A → T mutation destroys the start codon of the Cryge gene in the mutants; this mutation was confirmed by the absence of a restriction site for NcoI in the corresponding genomic fragment of homozygous mutants. The next in-frame start codon is 129 bp downstream; this predicted truncated γE-crystallin consists of 131 amino acids, resulting in a molecular mass of 14 kD. However, another open reading frame was observed just 19 bp downstream of the regular Cryge start codon, resulting in a protein of 119 amino acids and a calculated molecular weight of 13 kD. Western blot analysis using polyclonal antibodies against γ-crystallins or the novel Aey1-specific protein demonstrated the specific expression of the Aey1 protein in the cataractous lenses only; the truncated form of the γE-crystallin could not be detected. Therefore, it is concluded that the novel protein destroys the sensitive cellular structure of the eye lens.

In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations1. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper2, leads to a significant increase in the number of mouse models3 available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html ).

The G protein-coupled receptor 30 (GPR30) has been claimed as an estrogen receptor. However, the literature reports controversial findings and the physiological function of GPR30 is not fully understood yet. Consistent with studies assigning a role of GPR30 in the cardiovascular and metabolic systems, GPR30 expression has been reported in small arterial vessels, pancreas and chief gastric cells of the stomach. Therefore, we hypothesized a role of GPR30 in the onset and progression of cardiovascular and metabolic diseases. In order to test our hypothesis, we investigated the effects of a high-fat diet on the metabolic and cardiovascular profiles of Gpr30-deficient mice (GPR30-lacZ mice). We found that GPR30-lacZ female, rather than male, mice had significant lower levels of HDL along with an increase in fat liver accumulation as compared to control mice. However, two indicators of cardiac performance assessed by echocardiography, ejection fraction and fractional shortening were both decreased in an age-dependent manner only in Gpr30-lacZ male mice. Collectively our results point to a potential role of Gpr30 in preserving lipid metabolism and cardiac function in a sex- and age-dependent fashion. (C) 2014 Elsevier B.V. All rights reserved.

All oocytes from a patient who had undergone four unsuccessful in-vitro fertilization attempts showed neither a polar body nor pronuclei when examined for fertilization. In 19 inseminated oocytes that were spread for karyotypic analysis, one haploid set of metaphase II chromosomes and a remarkable condensed structure were found. Hormonal and morphological criteria implied that the oocytes had been mature at the time of retrieval. Since non-inseminated oocytes contained only one set of metaphase II chromosomes, the condensed structure appeared to represent the sperm chromatin in the state of premature chromosome condensation due to a block in oocyte maturation. Since the first and second polar body, as well as their chromatin, were undetectable in all the patient's oocytes, a rapid maturation to metaphase II before retrieval and prolonged arrest in this state before fertilization, accompanied by degeneration of the first polar body, appear to be responsible for the condition. In accordance with this notion, degenerate spindles (typical of post-ovulatory aged oocytes) and separating chromosomes (probably representing presegregating chromatids) were observed by antitubulin immunofluorescence.

Objective It is difficult to identify a single causative factor for inflammatory arthritis because of the multifactorial nature of the disease. This study was undertaken to dissect the molecular complexity of systemic inflammatory disease, utilizing a combined approach of mutagenesis and systematic phenotype screening in a murine model. Methods In a large-scale N-ethyl-N-nitrosourea mutagenesis project, the Ali14 mutant mouse strain was established because of dominant inheritance of spontaneous swelling and inflammation of the hind paws. Genetic mapping and subsequent candidate gene sequencing were conducted to find the causative gene, and systematic phenotyping of Ali14/+ mice was performed in the German Mouse Clinic. Results A novel missense mutation in the phospholipase Cγ2 gene (Plcg2) was identified in Ali14/+ mice. Because of the hyperreactive external entry of calcium observed in cultured B cells and other in vitro experiments, the Ali14 mutation is thought to be a novel gain-of-function allele of Plcg2. Findings from systematic screening of Ali14/+ mice demonstrated various phenotypic changes: an abnormally high T cell:B cell ratio, up-regulation of Ig, alterations in body composition, and a reduction in cholesterol and triglyceride levels in peripheral blood. In addition, spermatozoa from Ali14/+ mice failed to fertilize eggs in vitro, despite the normal fertility of the Ali14/+ male mice in vivo. Conclusion These results suggest that the Plcg2-mediated pathways play a crucial role in various metabolic and sperm functions, in addition to initiating and maintaining the immune system. These findings may indicate the importance of the Ali14/+ mouse strain as a model for systemic inflammatory diseases and inflammation-related metabolic changes in humans.

Multiple reports implicated the function of G protein-coupled receptor (GPR)-30 with nongenomic effects of estrogen, suggesting that GPR30 might be a G-protein coupled estrogen receptor. However, the findings are controversial and the expression pattern of GPR30 on a cell type level as well as its function in vivo remains unclear. Therefore, the objective of this study was to identify cell types that express Gpr30 in vivo by analyzing a mutant mouse model that harbors a lacZ reporter (Gpr30-lacZ) in the Gpr30 locus leading to a partial deletion of the Gpr30 coding sequence. Using this strategy, we identified the following cell types expressing Gpr30: 1) an endothelial cell subpopulation in small arterial vessels of multiple tissues, 2) smooth muscle cells and pericytes in the brain, 3) gastric chief cells in the stomach, 4) neuronal subpopulations in the cortex as well as the polymorph layer of the dentate gyrus, 5) cell populations in the intermediate and anterior lobe of the pituitary gland, and 6) in the medulla of the adrenal gland. In further experiments, we aimed to decipher the function of Gpr30 by analyzing the phenotype of Gpr30-lacZ mice. The body weight as well as fat mass was unchanged in Gpr30-lacZ mice, even if fed with a high-fat diet. Flow cytometric analysis revealed lower frequencies of T cells in both sexes of Gpr30-lacZ mice. Within the T-cell cluster, the amount of CD62L-expressing cells was clearly reduced, suggesting an impaired production of T cells in the thymus of Gpr30-lacZ mice.

PURPOSE: The purpose of the study was the characterization of the novel small-eye mutant Aey12 in the mouse. METHODS: The eyes of the mutants were described morphologically and histologically and by in situ hybridization. RESULTS: The homozygotes were viable and fully fertile, which identifies Aey12 as a new microphthalmia phenotype in the mouse, different from Maf or Pax6 mutants. Histologic analysis indicated the presence of the lens vesicle; however, the primary fiber cells did not elongate properly. Genome-wide linkage analysis mapped the mutation to the proximal region of chromosome 10 between the markers D10Mit206 and D10Mit189. Among the positional candidate genes, one EST (expressed sequence tag), D230044M03Rik, encodes a connexin-like protein. A G-->T point mutation was identified at cDNA position 96, resulting in an R32Q amino acid exchange in a transmembrane domain. The mutation leads to a loss of an SsiI restriction site, which is present in five wild-type mouse strains (102, C3H, C57BL/6, DBA, and JF1). The gene is expressed in the posterior part of the lens vesicle, where the primary fiber elongation starts. In the mutants, the expression pattern of Pax6, Prox1, Six3, and Crygd are modified, but not the pattern of Pax2. CONCLUSIONS: The mutated mouse gene belongs to the family of connexin-encoding genes (gene symbols Gja-Gje). Together with its rat and human homologues, it defines a new subgroup, referred to as Gjf1. The mouse mutant described herein offers a new functional candidate gene for microphthalmia-related disorders at the corresponding locus on human chromosome 6, area q24.

The Notch signaling pathway is an evolutionarily conserved transduction pathway involved in embryonic patterning and regulation of cell fates during development. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, which are also involved in distinct human diseases. Delta1 is one of the known ligands of the Notch receptors. Mice homozygous for a loss-of-function allele of the Delta1 gene Dll1lacZ/lacZ die during embryonic development. Here, we present the results of two phenotype-driven modifier screens. Heterozygous Dll1lacZ knockout animals were crossed with ENU-mutagenized mice and screened for dysmorphological, clinical chemical, and immunological variants that are dependent on the Delta1 loss-of-function allele. First, we show that mutagenized heterozygous Dll1lacZ offspring have reduced body weight and altered specific clinical chemical parameters, including changes in metabolites and electrolytes relevant for kidney function. In our mutagenesis screen we have successfully generated 35 new mutant lines. Of major interest are 7 mutant lines that exhibit a Dll1lacZ/+-dependent phenotype. These mutant mouse lines provide excellent in vivo tools for studying the role of Notch signaling in kidney and liver function, cholesterol and iron metabolism, cell-fate decisions, and during maturation of T cells in the immune system.

Background Primary cellular immunodeficiencies are a group of genetic disorders in which 1 or more components of the cellular immune system are lacking or dysfunctional. Objective We sought to identify novel mouse mutants that display primary cellular immunodeficiencies. Methods Genome-wide N-ethyl-N-nitrosourea mutagenesis was performed in mice, followed by a phenotype screen of immunologic blood parameters. Results We identified novel mouse mutants with isolated B-cell deficiency, combined block in early B- and T-cell development, combined T-cell and natural killer cell reduction, and 3 different forms of T-cell deficiencies. One of the mutants, designated ΔT3, displayed a combined phenotype of increased IgE, absence of peripheral T cells, and block in late thymocyte differentiation. In addition, ΔT3 mice were unable to mount specific humoral immune responses. Chromosomal mapping and sequencing of candidate genes revealed a novel point mutation in the kinase domain of the T-cell receptor ζ chain–associated protein kinase (Zap70). In contrast to Zap70-deficient mice, ΔT3 mutants displayed normal Zap70 mRNA and residual Zap70 protein levels. Complementation studies with Zap70-deficient mice confirmed that the point mutation found in Zap70 was causative for the ΔT3 phenotype, including increased IgE plasma levels, a phenotype that has not been associated with altered Zap70 function in the past. Conclusion Random genome-wide mutagenesis combined with a phenotype screen can be used to generate novel mouse mutants with primary cellular immunodeficiencies.

To detect mice with hereditary retinal impairment, a high-throughput electroretinography (ERG) screening system was established.Mice from eight different strains without known retinal disorders (102, 129/SvJ, AKR, C57BL/6J, C57BL/6JIco, CBA/CaJ, and DBA/2NCrlBR) and one control strain with retinal degeneration (C3HeB/FeJ) were fixed on a specially constructed sled, ERG electrodes were placed on the cornea, and mice were moved into a Ganzfeld stimulator. From a luminance range of 0.0125 to 500 cd-s/m(2) in a pretest series two levels (5 and 125 cd-s/m(2)) were chosen to shorten examination times. The root mean square (RMS) of the ERG-recording was analyzed to detect animals with abnormal retinal function. ERG responses of the left and right eyes were compared in amplitudes and implicit times of the a- and b-waves. Statistical analysis of the latter parameters was performed in all wild-type animals. Histology was performed on selected mice.ERG recordings of individual animals for the left and right eye revealed good agreement in amplitudes and implicit times of the a- and b-waves (P0.05). Comparison of these parameters among the wild-type strains showed several differences. Evaluation of the RMS revealed, in addition to the C3HeB/FeJ mice, a subgroup of mice within the 129/SvJ strain with abnormal retinal function. Molecular analysis of these mice demonstrated the presence of the same retroviral insertion in the Pde6b gene, which is causative of the Pde6b(rd1) allele carried in C3HeB/FeJ mice. Histologic analysis demonstrated good correlation between retinal electrophysiology and morphology.The present results demonstrate the feasibility of ERG for screening a large number of mice to detect animals with functional retinal impairment.

During a large-scale ENU mutagenesis screen, a mouse mutant with a dominant cataract was detected and referred to as Aey4. Aim of this study was the morphological description of the mutant, the mapping of the mutation, and the characterization of the underlying molecular lesion. The slit-lamp examination revealed a strong nuclear cataract surrounded by a homogeneous milky opacity in the inner cortex. The histological analysis demonstrated remnants of cell nuclei throughout the entire lens. The mutation was mapped to Chromosome 1 by a genome-wide linkage making the six gamma-crystallin encoding genes and the closely linked betaA2-crystallin encoding gene to relevant candidate genes. Finally, a T-->A exchange in exon 2 of the gammaD-crystallin encoding gene (symbol: Crygd) was demonstrated to be causative for the cataract phenotype; this particular mutation is, therefore, referred to Crygo(Aey4). The alteration in codon 76 leads to an amino acid exchange of Val-->Asp. Val at this position is highly conserved; it is found in all mouse and rat gammaD/E/F-crystallins as well as in the human gammaA- and gammaD-crystallins. It may be replaced solely by Ile, which is present in all bovine gamma-crystallins, in the rat and mouse gammaA/B/C-crystallins, as well as in the human gammaB/C-crystallins. It is predicted that the exchange of a hydrophobic side chain by a polar and acidic one might influence the microenvironment by a dramatic decrease of the isoelectric point by 1.5 pH units in the 10 amino acids surrounding position 76. The Crygd(Aey4) additionally demonstrates the importance of the integrity of the Cryg gene cluster for lens transparency.

During an ethylnitrosourea mutagenesis screen, Aey5 , a new mouse mutation exhibiting an autosomal dominant congenital cataract was isolated. The cataractous phenotype is visible at the eye opening and progresses to a nuclear and zonular cataract at 2 months of age with no difference in onset or severity between heterozygous and homozygous mutants. Histological analysis revealed that fiber cell differentiation continues at the lens bow region, but the cell nuclei do not degrade normally and remain in the deeper cortex. Further, the lens nucleus has clefts of various sizes while the remainder of the eye was morphologically normal. The mutation was mapped to chromosome 3 between the markers D3Mit101 and D3Mit77 near the connexin encoding genes Gja5 and Gja8 . Sequence analysis revealed no differences in the Gja5 gene, but identified a T→C mutation at position 191 in the Gja8 gene, which was confirmed by an additional Mva 12691 restriction site in the genomic DNA of homozygous mutants. This mutation results in Val→Ala substitution at codon 64 of connexin50 (Cx50) also known as lens membrane protein 70 (MP70). Aey5 represents the second dominant mouse cataract mutant affecting Cx50, a membrane protein preferentially expressed in the lens. Since both mutations affect similar regions in the first extracellular domain this region appears to be critically important for its function in lens transparency.

Recent in vivo and in vitro data of patients analyzed for genetic susceptibility to radiation during cancer therapy have shown structural changes in the chromosomes to be prevalent both in the patients being treated and in their immediate family members. As structural changes in chromosomes frequently lead to activation of proto-oncogenes and elimination of tumor-suppressor genes, they represent important mechanisms for the initiation of DNA repair processes and tumorigenesis. With the exception of rare genetic syndromes such as AT (Ataxia telangiectasia) or NBS (Nijmegen Breakage Syndrome), the background for the inheritance of genetic susceptibility to radiation is unknown. Recently, a large-scale genetic screen of mouse mutants has been established within the German Human Genome Project (Hrabe de Angelis and Balling 1998). The goal of this ENU (ENU: ethylnitrosourea) mutagenesis screen is the generation of mutant mice that will serve as animal models for human diseases and genetic susceptibility. In order to fully utilize the potential of a genetic screen of this magnitude, in which exploration for genes responsible for genomic instability and radiation sensitivity is to occur, it is necessary to establish a simple assay system that is amenable to automation. Hence, we are using the single-cell gel electrophoresis (comet assay) to detect mouse mutants that display a genetic susceptibility to ionizing radiation. We have established the analysis parameters in the comet assay which are currently used to detect radiation-sensitive mouse mutants and to control the variance within the mouse population in the ENU screen. The assay can be used to isolate genes that are responsible for DNA repair and radiation sensitivity in mouse and human.

The immunology screen focuses on the identification of novel gene products involved in the mammalian immune response and on the establishment of mouse models for immunological disorders. For this purpose, high throughput and semi-automated techniques were developed and optimized for low cost per sample and reproducibility. All assays are designed to be nonconsumptive and are based on peripheral blood or direct PCR amplification.

The Munich ENU Mouse Mutagenesis Screen is a large-scale mutant production, phenotyping, and mapping project. It encompasses two animal breeding facilities and a number of screening groups located in the general area of Munich. A central database is required to manage and process the immense amount of data generated by the mutagenesis project. This database, which we named MouseNet©, runs on a Sybase platform and will finally store and process all data from the entire project. In addition, the system comprises a portfolio of functions needed to support the workflow management of the core facility and the screening groups. MouseNet© will make all of the data available to the participating screening groups, and later to the international scientific community. MouseNet© will consist of three major software components: • Animal Management System (AMS) • Sample Tracking System (STS) • Result Documentation System (RDS) MouseNet© provides the following major advantages: • being accessible from different client platforms via the Internet • being a full-featured multi-user system (including access restriction and data locking mechanisms) • relying on a professional RDBMS (relational database management system) which runs on a UNIX server platform • supplying workflow functions and a variety of plausibility checks.

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.

During the mouse ENU mutagenesis screen, mice were tested for the occurrence of dominant cataracts. One particular mutant was discovered as a progressive opacity (Po). Heterozygotes show opacification of a superficial layer of the fetal nucleus, which progresses and finally forms a nuclear opacity. Since the homozygotes have already developed the total cataract at eye opening, the mode of inheritance is semidominant. Linkage analysis was performed using a set of genome-wide microsatellite markers. The mutation was mapped to chromosome 11 distal of the marker D11Mit242 (9.3 +/- 4.4 cM) and proximal to D11Mit36 (2.3 +/- 2.3 cM). This position makes the betaA3/A1-crystallin encoding gene Cryba1 an excellent candidate gene. Mouse Cryba1 was amplified from lens mRNA. Sequence analysis revealed a mutation of a T to an A at the second base of exon 6, leading to an exchange of Trp by Arg. Computer analysis predicts that the fourth Greek key motif of the affected betaA3/A1-crystallin will not be formed. Moreover, the mutation leads also to an additional splicing signal, to the skipping of the first 3 bp of exon 6, and finally to the deletion of the Trp residue. Both types of mRNA are present in the homozygous mutant lenses. The mutation will be referred to as Cryba1(po1). This particular mouse mutation provides an excellent animal model for a human congenital zonular cataract with suture opacities, which is caused by a mutation in the homologous gene.

PURPOSE. To characterize three new mouse small-eye mutants detected during ethylnitrosourea mutagenesis programs. METHODS. Three new mouse small-eye mutants were morphologically characterized, particularly by in situ hybridization. The mutations were mapped, and the candidate gene was sequenced. The relative amount of Pax6-specific mRNA was determined by real-time PCR. Reporter gene analysis used Crygf and Six3 promoter fragments in front of a luciferase gene and HEK293 cells as recipients. RESULTS. The new mutations-ADD4802, Aey11, and Aey 18-were mapped to chromosome 2; causative mutations have been characterized in Pax6 (Aey11: C→T substitution in exon 8, creating a stop codon just in front of the homeobox; ADD4802: G→A substitution at the beginning of intron 8 changes splicing and leads to an altered open reading frame and then to a premature stop codon; Aey18: G→A exchange in the last base of intron 5a leads also to a splice defect, skipping exons 5a and 6). Real-time PCR indicated nonsense-mediated decay in Pax6 Aey11 and Pax6 Aey18 mutants but not in Pax6 ADD4802 . This result is supported by the functional analysis of corresponding expression constructs in cell culture, where the Aeyl 1 and Aey18 alleles did not show a stimulation of the Six3 promotor or an inhibition of the Crygf promoter (as wild-type constructs do). However, the Pax6 ADD4802 allele stimulated both promoters. CONCLUSIONS. Together with functional analysis in a reporter gene assay and immunohistochemistry using Pax6 antibodies, it is suggested that the Pax6 Aey11 and Pax6 Aey18 alleles act through a loss of function, whereas ADD4802 represents a gain-of-function allele.