Production of knockout rats using ENU mutagenesis and a yeast-based screening assay

The rat is a widely used model in biomedical research and is often the preferred rodent model in many areas of physiological and pathobiological research. Although many genetic tools are available for the rat, methods to produce gene-disrupted knockout rats are greatly needed. In this study, we developed protocols for creating N-ethyl-N-nitrosourea (ENU)-induced germline mutations in several rat strains. F1 preweanling pups from mutagenized Sprague Dawley (SD) male rats were then screened for functional mutations in Brca1 and Brca2 using a yeast gap-repair, ADE2-reporter truncation assay. We produced knockout rats for each of these two breast cancer suppressor genes.     

Establishment of Cre/LoxP recombination system in transgenic rats

The rat has offered an important animal model in biomedical research including surgical procedure. However, advanced genetic manipulation has progressed less far in the rat than in the mouse. Here we report the Cre/LoxP transgenic rat system, demonstrating conditional chromosomal translocation both in the fertilization and adult stage, spatio-temporal gene controlling by catheter-based adenoviral gene transfer, and muscular fusion events in the limb transplant. Taking advantage of the larger body size of the rat than the mouse, this rat system provides a potential value to evaluate biomedical and therapeutic significance for gene therapy and regenerative medicine.     

The role of rats in functional genomics

Two hundred years of physiological and pharmaceutical studies and a decade of transgenic technology and genomic resources have made the laboratory rat a major model for biomedical research.     

Trace metal imaging with high spatial resolution: applications in biomedicine

New generations of analytical techniques for imaging of metals are pushing hitherto boundaries of spatial resolution and quantitative analysis in biology. Because of this, the application of these imaging techniques described herein to the study of the organization and dynamics of metal cations and metal-containing biomolecules in biological cell and tissue is becoming an important issue in biomedical research. In the current review, three common metal imaging techniques in biomedical research are introduced, including synchrotron X-ray fluorescence (SXRF) microscopy, secondary ion mass spectrometry (SIMS), and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). These are exemplified by a demonstration of the dopamine-Fe complexes, by assessment of boron distribution in a boron neutron capture therapy cell model, by mapping Cu and Zn in human brain cancer and a rat brain tumor model, and by the analysis of metal topography within neuromelanin. These studies have provided solid evidence that demonstrates that the sensitivity, spatial resolution, specificity, and quantification ability of metal imaging techniques is suitable and highly desirable for biomedical research. Moreover, these novel studies on the nanometre scale (e.g., of individual single cells or cell organelles) will lead to a better understanding of metal processes in cells and tissues.     

Handling the cotton rat for research

First used as an animal model of poliomyelitis in the late 1940s, the cotton rat is currently used in biomedical research for studies of human respiratory syncytial virus and filariasis. The author provides useful information relevant to the care of these research animals, including husbandry considerations, proper handling, and common laboratory procedures.     

Generating mutant rats using the Sleeping Beauty transposon system

The laboratory rat is an invaluable animal model for biomedical research. However, mutant rat resource is still limited, and development of methods for large-scale generation of mutants is anticipated. We recently utilized the Sleeping Beauty (SB) transposon system to develop a rapid method for generating insertional mutant rats. Firstly, transgenic rats carrying single transgenes, namely the SB transposon vector and SB transposase, were generated. Secondly, these single transgenic rats were interbred to obtain doubly-transgenic rats carrying both transgenes. The SB transposon was mobilized in the germline of these doubly-transgenic rats, reinserted into another location in the genome and heterozygous mutant rats were obtained in the progeny. Gene insertion events were rapidly and non-invasively identified by the green fluorescence protein (GFP) reporter incorporated in the transposon vector, which utilizes a polyA-trap approach. Mutated genes were confirmed by either linker ligation-mediated PCR or 3′-rapid amplification of cDNA ends (3′RACE). Endogenous expression profile of the mutated gene can also be visualized using the LacZ gene incorporated as a promoter-trap unit in the transposon vector. This method is straightforward, readily applicable to other transposon systems, and will be a valuable mutant rat resource to the biomedical research community.     

The emerging role for rat models in gene discovery

Rat models have been used for many decades to study physiological and pathophysiological mechanisms. Prior to the release of the rat genome and new technologies for targeting gene manipulation, the rat had been the underdog in the genomics era, despite the abundance of physiological data compared to the mouse. The overarching goal of biomedical research is to improve health and advance medical science. Translating human disease gene discovery and validation in the rat, through the use of emerging technologies and integrated tools and databases, is providing power to understand the genetics, environmental influences, and biology of disease. In this review we briefly outline the rat models, bioinformatics tools, and technologies that are changing the landscape of translational research. The strategies used to translate disease traits to genes to function, and, ultimately, to improve human health is discussed. Finally, our perspective on how rat models will continue to positively impact biomedical research is provided.     

Transgenic Modifications of the Rat Genome

The laboratory rat (R. norvegicus) is a very important experimental animal in several fields of biomedical research. This review describes the various techniques that have been used to generate transgenic rats: classical DNA microinjection and more recently described techniques such as lentiviral vector-mediated DNA transfer into early embryos, sperm-mediated transgenesis, embryo cloning by nuclear transfer and germline mutagenesis. It will also cover techniques associated to transgenesis such as sperm cryopreservation, embryo freezing and determination of zygosity. The availability of several technologies allowing genetic manipulation in the rat coupled to genomic data will allow biomedical research to fully benefit from the rat as an experimental animal.     

The Nile grass rat as a laboratory animal

Rodents are the subjects of the overwhelming majority of laboratory animal studies, and most laboratory rodents are nocturnal. The availability of a suitable diurnal rodent would provide a more effective animal model for biomedical research applicable to humans. The author describes several characteristics of the Nile grass rat that make this diurnal murid rodent an attractive laboratory animal.     

Identification of a rat model for usher syndrome type 1B by N-ethyl-N-nitrosourea mutagenesis-driven forward genetics

The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis to increase genetic variation in laboratory rats and identified a recessive mutant, named tornado, showing aberrant circling behavior, hyperactivity, and stereotypic head shaking. More detailed analysis revealed profound deafness due to disorganization and degeneration of the organ of Corti that already manifests at the onset of hearing. We set up a single nucleotide polymorphism (SNP)-based mapping strategy to identify the affected gene, revealing strong linkage to the central region of chromosome 1. Candidate gene resequencing identified a point mutation that introduces a premature stopcodon in Myo7a. Mutations in human MYO7A result in Usher syndrome type 1B, a severe autosomal inherited recessive disease that involves deafness and vestibular dysfunction. Here, we present the first characterized rat model for this disease. In addition, we demonstrate proof of principle for the generation and cloning of human disease models in rat using ENU mutagenesis, providing good perspectives for systematic phenotypic screens in the rat.     

An Instruction on the In Vivo Shell-Less Chorioallantoic Membrane 3-Dimensional Tumor Spheroid Model

The traditional shell chicken chorioallantoic membrane (CAM) model has been used extensively in cancer research to study tumor growth and angiogenesis. Here we present a combined in vivo tumor spheroid and shell-less CAM three-dimensional model for use in quantitative and qualitative analysis. With this model, the angiogenic and tumorigenic environments can be generated locally without exogenous growth factors. This physiological model offers a stable, static and flat environment that features a large working area and wider field of view useful for imaging and biomedical engineering applications. The short experimental time frame allows for rapid data acquisition, screening and validation of biomedical devices. The method and application of this shell-less model are discussed in detail, providing a useful tool for biomedical engineering research.     

Amplification of rat microsatellite loci in Mastomys coucha Smith, 1836

The multimammate rat M. coucha is the most widespread strain to be introduced in biomedical research and various stocks of this strain are maintained in laboratories across the globe. It is an ideal carrier of normally non-human disease to the domestic environment. In order to analyze genetic purity, strains of M. coucha were subjected to PCR-based DNA fingerprinting using sequence tagged microsatellite markers to evolve molecular signature to them. For this, 10 rats sequenced tagged microsatellite markers were used to investigate for their applicability of cross-species amplification in the genome of M. coucha. Out of 10 microsatellite primers tested, four (40%) microsatellite primer pairs [Carboxypeptidase B (CBP), Calmodulin (CALM3), Cell surface protein (CSPMO2) and Insulin like growth factor-I (IGF-1)] could be amplified successfully with exact with product size of 159, 145, 186 and 203 bps respectively in rat. The results suggest that since the above mentioned microsatellite primers get amplified successfully in M. coucha, they may be useful for genetic characterization, evaluation, strain improvement and biomedical research.     

长寿型啮齿类动物的抗肿瘤机制

啮齿类动物是广泛应用于生物医学的重要模式动物,包括先天性胸腺缺陷型的裸鼠、不患癌的裸鼹鼠(Heterocephalus glaber)和盲鼹鼠(Spalax galili)等。哺乳动物的衰老过程与癌症发生率有关,衰老的程度与患癌机率呈正相关。由于啮齿类动物约占哺乳动物的40%,因此研究长寿型啮齿类动物抗肿瘤机制对于抗癌机制的研究具有十分重要的作用。复制性衰老是啮齿类动物中普遍存在的抗肿瘤机制,但在裸鼹鼠和盲鼹鼠体内发现了独特的抗肿瘤机制：盲鼹鼠主要的抗肿瘤机制是由细胞释放IFN-β,激活p53和Rb信号通路,进而导致细胞集中性死亡;裸鼹鼠的抗肿瘤机制是由高分子量透明质酸引起的早期接触性抑制介导。此外,裸鼹鼠和盲鼹鼠的基因组中还含有高表达与调节细胞死亡和抗炎机制相关的基因。本文对裸鼹鼠和盲鼹鼠的独特抗肿瘤机制进行了综述,以期为该领域的相关研究提供参考。     

Regulation of hepatic glucose metabolism by leptin in pig and rat primary hepatocyte cultures

Direct effects of leptin on gluconeogenesis in rat hepatocytes are equivocal, and model systems from other species have not been extensively explored in assessing the regulation of glucose metabolism by leptin. Therefore, the goal of the present study was to compare the effects of leptin on gluconeogenesis in pig and rat hepatocyte cultures as well as to investigate an underlying mechanism of action at the level of phosphoenolpyruvate carboxykinase (PEPCK). In rat hepatocytes, leptin exposure (3 h, 50 and 100 nM) attenuated glucagon-stimulated hepatic gluconeogenesis by 35 and 38% (P < 0.05), respectively. However, leptin did not produce any significant acute effect in pig hepatocytes. Leptin exposure for 24 h failed to produce any significant effect on gluconeogenesis in either rat or pig hepatocytes cultured in the presence of glucagon or dexamethasone. Mechanistically, there was a 25-35% decrease (P < 0.05) in glucagon-induced PEPCK mRNA levels in rat but not pig hepatocytes cultured with leptin. This effect on PEPCK mRNA was not due to an alteration in the relative abundance of the leptin receptor or the ability of PEPCK to respond to cAMP. The nonuniformity of the effects of leptin on gluconeogenesis in pig and rat hepatocytes indicates differences in leptin action between species. Furthermore, the unique action of leptin in porcine hepatocytes points to the utility of this model system for biomedical research and also underscores the value of comparative studies.     

A joint latent variable model approach to item reduction and validation

Many applications of biomedical science involve unobservable constructs, from measurement of health states to severity of complex diseases. The primary aim of measurement is to identify relevant pieces of observable information that thoroughly describe the construct of interest. Validation of the construct is often performed separately. Noting the increasing popularity of latent variable methods in biomedical research, we propose a Multiple Indicator Multiple Cause (MIMIC) latent variable model that combines item reduction and validation. Our joint latent variable model accounts for the bias that occurs in the traditional 2-stage process. The methods are motivated by an example from the Physical Activity and Lymphedema clinical trial in which the objectives were to describe lymphedema severity through self-reported Likert scale symptoms and to determine the relationship between symptom severity and a "gold standard" diagnostic measure of lymphedema. The MIMIC model identified 1 symptom as a potential candidate for removal. We present this paper as an illustration of the advantages of joint latent variable models and as an example of the applicability of these models for biomedical research.     

Beyond the Rat Models of Human Neurodegenerative Disorders

The rat is a model of choice in biomedical research for over a century. Currently, the rat presents the best “functionally” characterized mammalian model system. Despite this fact, the transgenic rats have lagged behind the transgenic mice as an experimental model of human neurodegenerative disorders. The number of transgenic rat models recapitulating key pathological hallmarks of Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, or human tauopathies is still limited. The reason is that the transgenic rats remain more difficult to produce than transgenic mice. The gene targeting technology is not yet established in rats due to the lack of truly totipotent embryonic stem cells and cloning technology. This extremely powerful technique has given the mouse a clear advantage over the rat in generation of new transgenic models. Despite these limitations, transgenic rats have greatly expanded the range of potential experimental approaches. The large size of rats permits intrathecal administration of drugs, stem cell transplantation, serial sampling of the cerebrospinal fluid, microsurgical techniques, in vivo nerve recordings, and neuroimaging procedures. Moreover, the rat is routinely employed to demonstrate therapeutic efficacy and to assess toxicity of novel therapeutic compounds in drug development. Here we suggest that the rat constitutes a slightly underestimated but perspective animal model well-suited for understanding the mechanisms and pathways underlying the human neurodegenerative disorders.     

Effect of Cynomorium total flavone on depression model of perimenopausal rat

PurposeTo observe the effect of cynomorium total flavone on the depression model of perimenopausal rat and to analyze the action characteristics of cynomorium total flavone on depression of rat with perimenopausal syndrome.MethodDuplicate the model of rat with perimenopausal depression based on the combined method of incomplete castration and chronic stimulation, and keep drug administration for 35d. And then measure related behavior indicators and the change of biochemical index level in serum and brains; measure the estrogen/androgen receptor (ER/AR) in related tissues and the ERmRNA expression in hypothalamus.ResultIt can be seen that cynomorium total flavone can significantly improve the behavior indicators of rat with perimenopausal depression; obviously or significantly change the level of related biomedical indexes in serum and brains of perimenopausal depressed rat; obviously or significantly increase the expression of ER/AR in related tissues of perimenopausal depressed rat; obviously or significantly increase the ERmRNA expression in hypothalamus.ConclusionCynomorium total flavone can adjust hypothalamic-pituitary-gonadal axis by increasing E2, and make related biomedical indexes and hormone receptors tend to be normal, so as to relieve perimenopausal syndrome and perimenopausal syndrome with depression.