Current protocols in mouse biology最新文献

Diabetic retinopathy is a major cause of visual impairment, which continues to increase in prevalence as more and more people develop diabetes. Despite the importance of vision, the retina is one of the smallest tissues in the body, and specialized techniques have been developed to study retinopathy. This article summarizes several methods used to (i) induce diabetes in mice, (ii) maintain the diabetic animals throughout the months required for development of typical vascular histopathology, (iii) evaluate vascular histopathology of diabetic retinopathy, and (iv) quantitate abnormalities implicated in the development of the retinopathy.

Circadian rhythms take place with a periodicity of 24 hr, temporally following the rotation of the earth around its axis. Examples of circadian rhythms are the sleep/wake cycle, feeding, and hormone secretion. Light powerfully entrains the mammalian clock and assists in keeping animals synchronized to the 24-hour cycle of the earth by activating specific neurons in the "central pacemaker" of the brain, the suprachiasmatic nucleus. Absolute periodicity of an animal can deviate slightly from 24 hr as manifest when an animal is placed into constant dark or "free-running" conditions. Simple measurements of an organism's activity in free-running conditions reveal its intrinsic circadian period. Mice are a particularly useful model for studying circadian rhythmicity due to the ease of genetic manipulation, thus identifying molecular contributors to rhythmicity. Furthermore, their small size allows for monitoring locomotion or activity in their homecage environment with relative ease. Several tasks commonly used to analyze circadian periodicity and plasticity in mice are presented here including the process of entrainment, determination of tau (period length) in free-running conditions, determination of circadian periodicity in response to light disruption (e.g., jet lag studies), and evaluation of clock plasticity in non-24-hour conditions (T-cycles). Studying the properties of circadian periods such as their phase, amplitude, and length in response to photic perturbation, can be particularly useful in understanding how humans respond to jet lag, night shifts, rotating shifts, or other transient or chronic disruption of environmental surroundings.

Current comprehensive mouse metabolic phenotyping involves studying energy balance in cohorts of mice via indirect calorimetry, which determines heat release from changes in respiratory air composition. Here, we describe the measurement of daily energy expenditure (DEE) and basal metabolic rate (BMR) in mice. These well-defined metabolic descriptors serve as meaningful first-line read-outs for metabolic phenotyping and should be reported when exploring energy expenditure in mice. For further guidance, the issue of appropriate sample sizes and the frequency of sampling of metabolic measurements is also discussed.

Despite the dramatic increase in human lifespan over the past century, there remains pronounced variability in “health-span,” or the period of time in which one is generally healthy and free of disease. Much of the variability in health-span and lifespan is thought to be genetic in origin. Understanding the genetic mechanisms of aging and identifying ways to boost longevity is a primary goal in aging research. Here, we describe a pipeline of phenotypic assays for assessing mouse models of aging. This pipeline includes behavior/cognition testing, body composition analysis, and tests of kidney function, hematopoiesis, and immune function, as well as physical parameters. We also describe study design methods for assessing lifespan and health-span, and other important considerations when conducting aging research in the laboratory mouse. The tools and assays provided can assist researchers with understanding the correlative relationships between age-associated phenotypes and, ultimately, the role of specific genes in the aging process. © 2015 by John Wiley & Sons, Inc.

This article describes methodologies to examine mitochondrial respiration in fresh preparations of mouse tissues, including skeletal muscle, heart, liver, white and brown adipose tissue, and brain. Reference values and tips to maximize experimental efficiencies are also provided. Finally, correction methods and complementary techniques to properly interpret the results are presented and contrasted. © 2015 by John Wiley & Sons, Inc.

Cre-inducible mouse models are often utilized for the spatial and temporal expression of oncogenes. With the wide number of Cre recombinase lines available, inducible transgenesis represents a tractable approach to achieve discrete oncogene expression. Here, we describe a protocol for targeting Cre-inducible genes to the ubiquitously expressed ROSA26 locus. Gene targeting provides several advantages over standard transgenic techniques, including a known site of integration and previously characterized pattern of expression. Historically, an inherent instability of ROSA26 targeting vectors has hampered the efficiency of developing ROSA26 knock-in lines. In this protocol, we provide individual steps for utilizing Gateway recombination for cloning as well as detailed instructions for screening targeted ES cell clones. By following this protocol, one can achieve germline transmission of a ROSA26 knock-in line within several months. © 2015 by John Wiley & Sons, Inc.

Diabetic nephropathy (DN) is a term used to describe kidney damage cause by diabetes. With DN as one of the leading causes of end-stage renal disease worldwide, there is a strong need for appropriate animal models to study DN pathogenesis and develop therapeutic strategies. To date, most experiments are carried out in mouse models as opposed to other species for several reasons including lower cost, ease of handling, and easy manipulation of the mouse genome to generate transgenic and knockout animals. This unit provides detailed insights and technical knowledge in setting up one of the most widely used models of DN, the streptozotocin (STZ)-induced model. This model has been extensively exploited to study the mechanism of diabetic renal injury. The advantages and limitations of the STZ model and the availability of other genetic models of DN are also discussed. © 2015 by John Wiley & Sons, Inc.

Haploid genetics has facilitated new insights into mammalian pathways and disease mechanisms. Most animal cells are diploid, and mammalian haploid cell cultures have remained elusive for a long time. Recent methodological progress has enabled the routine derivation of haploid stem cell lines from mammalian haploid embryos. Here we provide detailed protocols for the establishment, culture, and manipulation of parthenogenetic and androgenetic haploid embryonic stem cells from mouse embryos. © 2015 by John Wiley & Sons, Inc.

Three commonly used murine surgical models of bone healing [closed fracture with intramedullary fixation, distraction osteogenesis (DO), and marrow ablation by reaming] are presented. Detailed surgical protocols for each model are outlined. The nature of the regenerative processes and the types of research questions that may be addressed with these models are briefly outlined. The relative strengths and weaknesses of these models are compared to a number of other surgical models that are used to address similar research questions. © 2015 by John Wiley & Sons, Inc.

Providing anesthesia and analgesia for mouse subjects is a common and critical practice in the laboratory setting. These practices are necessary for performing invasive procedures, achieving prolonged immobility for sensitive imaging modalities (magnetic resonance imaging for instance), and providing intra- and post-procedural pain relief. In addition to facilitating the procedures performed by the investigator, the provision of anesthesia and analgesia is crucial for the preservation of animal welfare and for humane treatment of animals used in research. Furthermore, anesthesia and analgesia are important components of animal use protocols reviewed by Institutional Animal Care and Use Committees, requiring careful consideration and planning for the particular animal model. In this article, we provide technical outlines for the investigator covering the provision of anesthesia by two routes (injectable and inhalant), guidelines for monitoring anesthesia, current techniques for recognition of pain, and considerations for administering preventative analgesia. © 2015 by John Wiley & Sons, Inc.