Despite the pre-eminence of the mouse in modelling human disease, several aspects of murine biology limit its routine use in large-scale genetic and therapeutic screening. Many researchers who are interested in an embryologically and genetically tractable disease model have now turned to zebrafish. Zebrafish biology allows ready access to all developmental stages, and the optical clarity of embryos and larvae allow real-time imaging of developing pathologies. Sophisticated mutagenesis and screening strategies on a large scale, and with an economy that is not possible in other vertebrate systems, have generated zebrafish models of a wide variety of human diseases. This Review surveys the achievements and potential of zebrafish for modelling human diseases and for drug discovery and development.

Animal models of human disease: zebrafish swim into view.

■ Abstract Phenotypic variation for quantitative traits results from the segregation of alleles at multiple quantitative trait loci (QTL) with effects that are sensitive to the genetic, sexual, and external environments. Major challenges for biology in the post-genome era are to map the molecular polymorphisms responsible for variation in medically, agriculturally, and evolutionarily important complex traits; and to determine their gene frequencies and their homozygous, heterozygous, epistatic, and pleiotropic effects in multiple environments. The ease with which QTL can be mapped to genomic intervals bounded by molecular markers belies the difficulty in matching the QTL to a genetic locus. The latter requires high-resolution recombination or linkage disequilibrium mapping to nominate putative candidate genes, followed by genetic and/or functional complementation and gene expression analyses. Complete genome sequences and improved technologies for polymorphism detection will greatly advance the genetic dissection of quantitative traits in model organisms, which will open avenues for exploration of homologous QTL in related taxa.

The genetic architecture of quantitative traits

During the past decade, the molecular mechanisms underlying the mammalian circadian clock have been defined. A core set of circadian clock genes common to most cells throughout the body code for proteins that feed back to regulate not only their own expression, but also that of clock output genes and pathways throughout the genome. The circadian system represents a complex multioscillatory temporal network in which an ensemble of coupled neurons comprising the principal circadian pacemaker in the suprachiasmatic nucleus of the hypothalamus is entrained to the daily light/dark cycle and subsequently transmits synchronizing signals to local circadian oscillators in peripheral tissues. Only recently has the importance of this system to the regulation of such fundamental biological processes as the cell cycle and metabolism become apparent. A convergence of data from microarray studies, quantitative trait locus analysis, and mutagenesis screens demonstrates the pervasiveness of circadian regulation in biological systems. The importance of maintaining the internal temporal homeostasis conferred by the circadian system is revealed by animal models in which mutations in genes coding for core components of the clock result in disease, including cancer and disturbances to the sleep/wake cycle.

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Mammalian circadian biology: elucidating genome-wide levels of temporal organization.

Mice provide an experimental model of unparalleled flexibility for studying mammalian diseases. Inbred strains of mice exhibit substantial differences in their susceptibility to the renal complications of diabetes. Much remains to be established regarding the course of diabetic nephropathy (DN) in mice as well as defining those strains and/or mutants that are most susceptible to renal injury from diabetes. Through the use of the unique genetic reagents available in mice (including knockouts and transgenics), the validation of a mouse model reproducing human DN should significantly facilitate the understanding of the underlying genetic mechanisms that contribute to the development of DN. Establishment of an authentic mouse model of DN will undoubtedly facilitate testing of translational diagnostic and therapeutic interventions in mice before testing in humans.

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Mouse Models of Diabetic Nephropathy

Molecules that can specifically silence gene expression are powerful research tools. Much effort has been put into the development of such molecules and has resulted in the creation of different classes of potential therapeutic agents. Small interfering RNA (siRNA) is one of the latest additions to the repertoire of sequence-specific gene-silencing agents. The robustness of this approach has motivated numerous biotechnology organizations and academic institutions to develop siRNA libraries for high-throughput genome-wide screening in mammalian cells. This article first overviews current nucleic-acid-based approaches for gene silencing, and then focuses on the application of siRNAs in particular in functional genomics and as potential therapeutics.

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siRNAs: applications in functional genomics and potential as therapeutics.

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
 ).

Genome-wide, large-scale production of mutant mice by ENU mutagenesis

Since the mouse has become the most detailed model system to investigate the genetics and pathogenesis of human diseases, large numbers of new mouse strains have and continue to be produced. In nearly all animal facilities, the maintenance of breeding colonies is limited and mouse strains have to be archived in an efficient way. This study was undertaken to test the reliability of recovering mouse lines by use of cryopreserved spermatozoa from individual male mice. In contrast to many studies, spermatozoa and oocytes were derived from the same genetic background. 30 C3HeB/FeJ males belonging to three different categories (wild-type, F1-generation of ENU-treated males, and defined mutants) were recovered by producing at least 20 offspring from each donor. Independent of the experimental group, every single male was successfully recovered. Archiving mouse strains by cryopreservation of spermatozoa may, therefore, offer a reliable way to preserve genetically valuable mouse strains and provides an efficient management strategy for animal facilities.

Reliable recovery of inbred mouse lines using cryopreserved spermatozoa.

With the increasing demand for and production of transgenic and mutant mice for biomedical research, embryo transfer plays a paramount role The purpose of performing embryo transfer in this species is to generate transgenic mice via blastocyst injection of embryonic stem cells or pronuclear injection of DNA constructs, to revitalize cryopreserved sperm and embryos, and to generate mouse lines that meet specific pathogen-free health standards for breeding in barrier areas (rederivation) We present results from two years of carrying out embryo transfers for rederivation purposes in the large mouse breeding facility of the GSF—National Research Center for Environment and Health, Neuherberg, Germany Pathogens to be eradicated from inbred transgenic (C57BL/6 background) and mutant (C3H background) mouse lines included mouse hepatitis virus, mouse minute virus, and mouse parvovirus In vitro- and in vivo-produced two-cell embryos were washed 3 times in M2 medium A total of 20 embryos each were transferred to the oviduct of 8- to 12-week-old specific pathogen-free pseudopregnant (Day 05) Swiss recipients under aseptic conditions Mice were then kept singly in individually ventilated cages and manipulated in a Class II laminar flow hood From each transfer to one to five recipients with embryos originating from the same mouse line, one recipient was tested for the presence of microorganisms 6 to 12 weeks after embryo transfer, ie at 0 to 6 weeks after weaning, according to the FELASA (Federation of European Laboratory Animal Science Associations) Guidelines A total of 290 embryo transfers were performed for revitalization of cryopreserved sperm from 52 mouse lines, cryopreserved two-cell embryos from 18 mouse lines and rederivation of 12 mouse lines using freshly collected two-cell embryos From these 290 embryo transfers, 59 mouse lines were re-established (40 from cryopreserved sperm, 7 from cryopreserved embryos and 12 from in vivo-produced embryos) Health monitoring of 54 recipients showed that all mouse lines generated were free of all pathogens stated in the FELASA list The results presented here show that all 12 (100%) mouse lines were re-established after transfer of freshly collected two-cell embryos whereas 77% and 39% success rates were observed for revitalization of cryopreserved sperm and embryos, respectively The success of embryo transfer in eradicating pathogens depends on the inability of these pathogens to transverse the zona pellucida and enter and/or infect embryonic cells In our mouse facility, embryo transfer provided an efficient method to successfully revitalize cells of the mouse germ line as well as to eradicate prevalent murine pathogens Furthermore, the results demonstrate the efficiency of transferring embryos of different origins and thereby obtaining and maintaining specific pathogen-free health standards in our mouse colonies

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S. Marschall

Papers

Genome-wide, large-scale production of mutant mice by ENU mutagenesis

Reliable recovery of inbred mouse lines using cryopreserved spermatozoa.

179Importance of embryo transfers in transgenic mouse facilities