http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_10 Somatic cell nuclear transfer (SCNT) is a powerful tool for the investigation of the mechanisms of nuclear remodeling. In addition, SCNT may offer the possibility of introducing targeted mutations by homologous recombination in species for which ES cell technology is not available. The rat specific features of the oocyte have long impeded the development of SCNT. We detail here the procedures developed and optimized during the last several years for the optimization of rat cloning. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_11 The rat is one of the most preferred model organisms in biomedical research and has been extremely useful for linking physiology and pathology to the genome. However, approaches to genetically modify specific genes in the rat germ line remain relatively scarce. To date, the most efficient approach for generating genetically modified rats has been the target-selected N-ethyl-N-nitrosourea (ENU) mutagenesis-based technology. Here, we describe the detailed protocols for ENU mutagenesis and mutant retrieval in the rat model organism. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_12 Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. Their embryonic stem (ES) cells, after differentiation into each tissue or organ, are applied in regenerative medicine, which enables examination of the effects of drugs for various diseases. Knockout rats will also provide a suitable model system for many human diseases and a great amount of new insights into gene functions, which have not been revealed by knockout mice. In 2008, we experienced the world’s first success in establishing rat ES cells with chimeric contribution. Following on the heels of our report, others reported the establishment of rat ES cells that could complete a germline transmission. Recent studies on rat as well as mouse ES cells suggest that modifications of signal inhibitors and serum in the medium are critical for the maintenance of the pluripotency of ES cells. In this chapter, we discuss techniques for the successful establishment and maintenance of rat ES cells. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_13 Embryonic stem (ES) cells have been used extensively for site-specific gene targeting in the mouse. The resulting knock-out and knock-in mouse models generated so far have demonstrated their usefulness in biomedical research. However, for many diseases and fields of study, the rat still represents a superior model. The derivation and culture of germline-competent ES cells in the rat would allow the application of site-specific gene targeting technologies to this species of indisputable importance to biomedical research. We have recently shown the derivation, culture, and for the first time, in vivo contribution of rat ES-like cells to developing tissues. This represents an important step forward in making gene targeting technologies available to the rat research community, via development of rat ES cells. Here, we describe the materials, methods and techniques that have been used to obtain rat blastocysts, derive and culture embryonic cell lines from these, and assess the developmental capacity of the cells in vivo. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_14 With the lack of tools available to manipulate the rat genome, alternative technologies have been investigated to generate loss-of-function rat models by gene invalidation. The recent demonstration that RNA interference (RNAi)-mediated gene silencing occurs in rodents has opened new opportunities for rat functional genetics. In this chapter, we provide some practical guidelines for RNAi working in rat, based on the recent design and development of mice and rat Knock down models. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_15 The genetic dissection of physiological and pathological traits in laboratory model organisms is accelerated by the ability to engineer loss-of-function mutations at investigator-specified loci. This chapter describes the use of zinc-finger nucleases (ZFNs) for the targeted disruption of endogenous rat genes directly in the embryo. ZFNs can specifically disrupt target genes in cultured rat cells and in embryos from inbred and outbred strains, leading to permanently genetically modified animals. This technology allows for the rapid, targeted modification of the rat genome. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_16 The field of toxicogenomics, which is becoming an important sub-discipline of toxicology, resulted from the natural convergence of the field of conventional toxicological research and the emergent field of functional genomics. One technology that has played a significant role in the field of toxicogenomics (in addition to many others) is the gene expression microarray. In this chapter, the authors provide an example of the application of gene expression microarrays to the field of toxicogenomics by detailing the strategy that was used for obtaining, analyzing, and interpreting gene expression data generated from RNA isolated from the liver of toxicant-exposed rats. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_17 Congenic and consomic rat strains are inbred strains containing in their genome a given genomic region (congenic) or a whole chromosome (consomic) from another strain. They are nowadays invaluable tools for the identification of genes and mechanisms of multifactorial diseases, one of the main goals in biomedicine. They are produced by repeated backcrosses from a donor inbred strain to a recipient inbred strain, and thereafter maintained by conventional brother-x-sister mating. Although their production is lengthy and costly, it only requires a zootechny unit for breeding and tools for genotyping. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_18 Congenic strains are routinely used for positional mapping of quantitative trait loci; while conplastic strains, derived by substitution of different mitochondrial genomes on the same nuclear genetic background of inbred rodent strains, provide a way to unambiguously isolate effects of the mitochondrial genome on complex traits. Derivation of congenic or conplastic strains using a traditional backcross breeding strategy (10 backcrosses) takes more than 3 years. There are two principal strategies to speed up this process: (1) marker-assisted derivation of “speed” congenic/conplastic strains and (2) derivation of “supersonic” congenic/conplastic strains using in each backcross generation embryos obtained from 4-week-old superovulated females; thus, each backcross generation takes only 7 weeks. Both strategies could also be combined. In the current chapter, a method for derivation of “supersonic” congenic/conplastic rat strains is described. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_19 It is extremely useful to define a rapid and accurate method for identifying homozygous and heterozygous transgenic animals prior to setting up breeding programs for transgenic colonies and in experiments in which gene dosage effects could have a functional impact. Southern-blotting is a means of identifying zygosity, but such a method is time consuming and produces a high level of ambiguous results. Some years ago, we described the rapid, precise, non-ambiguous, and high-throughput identification of zygosity in transgenic animals by real-time PCR. This technique allows us to make a clear-cut identification of transgenic rats, transgenic mice, and double-transgenic pigs. Since 2002, however, several authors have made improvements to this method. The following paper describes the ease with which zygosity is determined using real-time PCR. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_1 The rat evokes fear and disgust in a large percentage of people around the world. Yet, other people are fascinated by this amazing creature that is raised as a pet, has an important place in several religions, and is a prominent model for biomedical research. This book focuses on a variety of methodologies that can be used in this remarkable model. This chapter sets the stage by providing a perspective on why the rat remains an important model in biomedical research. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_20 Currently, most genetically engineered rat strains are created by methods that involve random integration of transgenes into the genome. The ability to identify the chromosomal location of the transgene insertion site enables the development of efficient genotyping assays, allows segregation of multiple transgene integration sites to be followed while breeding, and facilitates characterization of possible positional effects on phenotype. Here we describe a method for determining the chromosomal location of transgene insertion that combines restriction endonuclease enzyme digest with subsequent rounds of PCR amplification to produce amplicons representing the chromosomal regions flanking the integrated transgene. This method provides a reliable means for determining the exact location of insertion of transgenes within the genome. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_21 The number of rat strains increased considerably in the last decade and will increase continuously during the next years. This requires enough space for maintaining vital strains and techniques for cryobanking, which can be applied not only in specialised rat ressource centres but also in regular animal houses. Here we describe an easy and fast method for the cryopreservation and transplantation of frozen-thawed ovaries of the rat. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_22 Although in vitro and in vivo fertilization are powerful tools for restoring conserved sperm as well as stocked males in the rat, the techniques have progressively gained importance. However, the techniques are not used extensively for efficient production of rat offspring, because the techniques require a great deal of skill. This chapter describes the protocols for in vitro and in vivo fertilization in the rat. Namely, sperm collection, sperm cryopreservation, pre-incubation of sperm, and insemination (co-culture with sperm and oocytes) for in vitro fertilization and intrauterine insemination for in vivo fertilization with fresh or frozen/thawed spermatozoa are provided. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_23 More than 500 inbred rat strains have been developed during the past 100 years for a wide range of biomedical applications. In addition to these traditionally bred strains, many induced mutants and several thousand mutagenized sperm samples have recently been generated. At present this huge number of strains is mainly managed by two rat resource centers, the National Bio Resource Project for the Rat in Japan (NBRP-Rat) and the US based Rat Resource and Research Center (RRRC). These resource centers not only collect, maintain and distribute rat strains as animals or cryopreserved embryos and spermatozoa, but also perform additional tasks such as phenotypic and genetic characterization as well as microbiological cleaning. Furthermore, they support researchers through informative databases in the selection of rat strains for specific research purposes. These global rat resource centers are essential for successful and sustainable research using the rat as a model species. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_24 Each translational approach in medical research forces the establishment of neurobehavioral screening systems, dedicated to fill the gap between postgenomic generation of state-of-the-art animal models (i.e. transgenic rats) on the one hand and their added value for really predictive experimental preclinical therapy on the other. Owing to these developments in the field, neuroscientists are frequently challenged by the task of detecting discrete behavioral differences in rats. Systematic, comprehensive phenotyping covers these needs and represents a central part of the process. In this chapter, we provide an overview on theoretical issues related to comprehensive neurobehavioral phenotyping of rats and propose specific classical procedures, protocols (similar to the SHIRPA approach in mice), as well as techniques for repeated, intraindividual phenotyping. Neurological testing of rats, motorfunctional screening using the accelerod approach, emotional screening using the social interaction test of anxiety, and testing of sensorimotoric gating functions by prepulse inhibition of the startle response are provided in more detail. This description is completed by an outlook on most recent developments in the field dealing with automated, intra-home-cage technologies, allowing continuous screening in rats in various behavioral and physiological dimensions on an ethological basis. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_25 Psychiatric diseases are very debilitating and some of them highly prevalent (e.g., depression or anxiety). The rat remains one model of choice in this discipline to investigate the neural mechanisms underlying normal and pathological traits. Genomic tools are now applied to identify genes involved in psychiatric illnesses and also to provide new biomarkers for diagnostic and prognosis, new targets for treatment and more generally to better understand the functioning of the brain. In this report, we will review rat models, behavioral approaches used to model psychiatry-related traits and the major studies published in the field including genetic mapping of quantitative trait loci (QTL), transcriptomics, proteomics and transgenic models. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_26 LEW and BN rats, that behave in opposite ways for their susceptibility to various immune-mediated diseases, provide a powerful model to investigate the molecular and genetic bases of immune system physiology and dysregulation. Using this model, we addressed the question of the genetic control of central nervous system autoimmunity, of xenobiotic-induced allergic diseases, and of T cell subsets that differ by their cytokine profiles. By linkage analysis and genetic dissection, using a panel of congenic rats, we identified a 120 Kb region on chromosome 9 that controls all these phenotypes, indicating that this region contains a gene or set of genes that plays an important role in the immune system homeostasis and susceptibility to immune mediated diseases. In this review, we will describe these rat genomics studies and will discuss the cellular and genetic factors that may be involved in the differences between these rat strains. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_27 In cardiovascular research, the rat has been the main model of choice for decades. Experimental procedures were developed to generate cardiovascular disease states in this species, such as systemic and pulmonary hypertension, cardiac hypertrophy and failure, myocardial infarction, and stroke. Furthermore, rats have been bred, which spontaneously develop such diseases. They became extremely valuable models to understand the genetics of these diseases, since powerful genomic tools are now available for the rat. One of these tools is transgenic technology, which has allowed the creation of even more disease models in the rat. This review summarizes the experimental, genetic, and transgenic rat models for cardiovascular diseases. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_28 The spontaneously hypertensive rat (SHR) is the most widely used animal model of essential hypertension and accompanying metabolic disturbances. In this model, the use of whole genome sequencing and gene expression profiling techniques, linkage and correlation analyses in recombinant inbred strains, and in vitro and in vivo functional studies in congenic and transgenic lines has recently enabled molecular identification of quantitative trait loci (QTLs) relevant to the metabolic syndrome: (1) a deletion variant in Cd36 (fatty acid translocase) responsible for QTLs on chromosome 4 associated with dyslipidemia, insulin resistance and hypertension, (2) mutated Srebf1 (sterol regulatory element binding factor 1) as a QTL on chromosome 10 influencing dietary-induced changes in hepatic cholesterol levels, and (3) Ogn (osteoglycin) as a QTL on chromosome 17 associated with left ventricular hypertrophy. In addition, selective replacement of the mitochondrial genome of the SHR with the mitochondrial genome of the Brown Norway rat influenced several major metabolic risk factors for type 2 diabetes and provided evidence that spontaneous variation in the mitochondrial genome per se can promote systemic metabolic disturbances relevant to the pathogenesis of metabolic syndrome. Owing to recent progress in the development of rat genomic resources, the pace of QTL identification and discovery of new disease mechanisms can be expected to accelerate in the near future. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_29 Current understanding of the mechanisms underlying renal disease in humans is incomplete. Consequently, our ability to prevent the occurrence of renal disease or treat kidney disease once it develops is limited. There are objective difficulties in investigating kidney disease directly in humans, leading investigators to resort to experimental animal models that simulate renal disease in humans. Animal models have thus been a tool of major importance in the study of normal renal physiology and have been crucial in shedding light on the complex mechanisms involved in normal kidney function and in our current understanding of and ability to treat renal disease. Among the animal models, rat has been the preferred and most commonly used species for the investigation of renal disease. This chapter reviews what has been achieved over the years, using rat as a tool for the investigation of renal disease in humans, focusing on the contribution of rat genetics and genomics to the elucidation of the mechanisms underlying the pathophysiology of the major types of renal disease, including primary and secondary renal diseases. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_2 Genetic mapping and positional cloning of genetically complex traits in the laboratory rat (Rattus norvegicus) has recently led to the identification of various susceptibility genes in different rat models. Rat genetics has benefited from revolutionary advances in molecular biology, genetics, genomics and informatics and provide an unparalleled resource for molecular genetic investigation of mammalian physiopathology and its underlying complex genetic architecture. In this review, we will consider different strategies that are being used in the successful positional cloning of rat complex trait genes in the context of recent progress in rodent and human genetics. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_30 Rat has been the major model species used in several biomedical fields, notably in drug development and toxicology, including carcinogenicity testing. Rat is also a useful model in basic cancer research. Several rat models of monogenic (Mendelian) human hereditary cancers are available. Some were obtained spontaneously, while others were generated either by mutagenesis of tumor suppressor genes or by transgenesis of activated oncogenes (transgenesis can be performed efficiently in the rat). In addition, among the hundreds of inbred rat strains that have been isolated, some are highly susceptible or resistant to certain types of cancer, and these divergent phenotypes were shown to be polygenic. Numerous quantitative trait loci (QTLs) controlling cancer susceptibility/resistance have been defined in linkage analyses, and several of these QTLs were physically demonstrated in congenic strains. These studies led, in particular, to rapid translation to the human, with the identification of loci controlling susceptibility to a form of multiple endocrine neoplasia (monogenic trait) and to breast cancer (polygenic disease). The biology of cancer resistance has also been analyzed, and in some (but not all) cases, it was linked to regression of preneoplasic lesions. Rat tumors have been the subject of various types of analyses, and these studies led to important conclusions, including that tumors can be classified on the basis of the identity of the inducing agent, thereby suggesting that analyses of human tumors may be valuable in determining retrospectively the role of specific carcinogens in the formation of human cancers, and of human breast cancer in particular. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_3 The rat is an important system for modeling human disease. Four years ago, the rich 150-year history of rat research was transformed by the sequencing and annotation of the rat genome, ushering in an era of exceptional opportunity for identifying genes and pathways underlying disease phenotypes. With the genome sequence in place, there is the prospect of not only linking the extensive literature of mechanistic and pharmacological studies in the rat to its genome, but by using comparative genomics to other organisms as well. Genome-wide association studies (GWAS) in human populations have recently provided a direct approach for finding robust genetic associations in common diseases, but identifying the precise genes and their mechanisms of action remains challenging. 2010-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-389-3_4 The use of transgenesis is relatively rare in rats, and this is because of the relative difficulty in adding foreign genes by the conventional methods. Gene knock out and knock in by the conventional techniques of homologous recombination remain difficult in rats. This situation would be less crucial if the gene constructs were more reliable for the expression of foreign genes. The present chapter describes the state of the art in vector design for various genetic modifications in rats. 2010-01-01T05:00:00Z