The dark phase improves genetic discrimination for some high throughput mouse behavioral phenotyping

Dark-phase testing has previously been shown by others to improve the outcome of some 'classical' behavior test situations. However, the importance of such ethological correctness and the effect of the light/dark cycle on high throughput behavioral testing situations such as 'mutant vs. wild type' and 'screening', are less or unknown, respectively. These testing situations differ from the 'classical' in that they are designed primarily to discriminate between genetically different mice rather than provide a detailed assessment of ability or psychosocial state. Here we test the hypotheses that dark-phase testing affects the outcome of high throughput behavioral tests and that dark-phase testing improves discrimination between genetically distinct mice (C57BL/6J, 129S1/SvImJ and B6129F1) using high throughput behavioral tests. Our results demonstrate that, although all successful tests showed some effect of phase, only the SHIRPA primary screen, open-field test and motor learning on the rotarod showed improved strain discrimination in the dark phase. Surprisingly, the social interaction test did not show a clear benefit to either phase, and interestingly, the tail-flick test discriminated strains better in the light phase. However, since the preponderance of our data shows that dark-phase testing improves, or does not affect, strain discrimination, we conclude that for these strains and tests, dark-phase testing provided superior outcomes. If discrimination is not achieved in the dark phase, then light phase-testing would be undertaken.     

Comparative genetic analysis: the utility of mouse genetic systems for studying human monogenic disease

One of the long-term goals of mutagenesis programs in the mouse has been to generate mutant lines to facilitate the functional study of every mammalian gene. With a combination of complementary genetic approaches and advances in technology, this aim is slowly becoming a reality. One of the most important features of this strategy is the ability to identify and compare a number of mutations in the same gene, an allelic series. With the advent of gene-driven screening of mutant archives, the search for a specific series of interest is now a practical option. This review focuses on the analysis of multiple mutations from chemical mutagenesis projects in a wide variety of genes and the valuable functional information that has been obtained from these studies. Although gene knockouts and transgenics will continue to be an important resource to ascertain gene function, with a significant proportion of human diseases caused by point mutations, identifying an allelic series is becoming an equally efficient route to generating clinically relevant and functionally important mouse models.     

Prostaglandin E synthases: Understanding their pathophysiological roles through mouse genetic models

Prostaglandin E synthase (PGES), which converts cyclooxygenase (COX)-derived prostaglandin H₂ (PGH₂) to PGE₂, is known to comprise a group of at least three structurally and biologically distinct enzymes. Two of them are membrane-bound and have been designated as mPGES-1 and mPGES-2. mPGES-1 is a perinuclear protein that is markedly induced by proinflammatory stimuli and downregulated by anti-inflammatory glucocorticoids as in the case of COX-2. It is functionally coupled with COX-2 in marked preference to COX-1. mPGES-2 is synthesized as a Golgi membrane-associated protein, and the proteolytic removal of the N-terminal hydrophobic domain leads to the formation of a mature cytosolic enzyme. This enzyme is rather constitutively expressed in various cells and tissues and is functionally coupled with both COX-1 and COX-2. Cytosolic PGES (cPGES) is constitutively expressed in a wide variety of cells and is functionally linked to COX-1 to promote immediate PGE₂ production. Recently, mice have been engineered with specific deletions in each of these three PGES enzymes. In this review, we summarize the current understanding of the in vivo roles of PGES enzymes by knockout mouse studies and provide an overview of their biochemical properties.     

Defining the phenotype in human genetic studies: forward genetics and reverse phenotyping

The definition of phenotypes for genetic study is a challenging endeavor. Just as we apply strict quality standards to genotype data, we should expect that phenotypes meet consistently high standards of reproducibility and validity. The methods for achieving accurate phenotype assignment in the research setting--the 'research diagnosis'--are different from the methods used in clinical diagnosis in the patient care setting. We evaluate some of the main challenges of phenotype definition in human genetics, and begin to outline a set of standards to which phenotypes used in genetics studies may aspire with the goal of increasing the quality and reproducibility of linkage and association studies. Revisiting the traditional phenotype definitions through a focus on familial components and heritable endophenotypes is a time-honored approach. Reverse phenotyping, where phenotypes are refined based on genetic marker data, may be a promising new approach. The stakes are high, since the success of gene mapping in genetically complex disorders hinges on the ability to delineate the target phenotype with accuracy and precision.     

Novel throughput phenotyping platforms in plant genetic studies

Unraveling the genetic basis of complex traits in plants is limited by the lack of appropriate phenotyping platforms that enable high-throughput screening of many genotypes in multilocation field trials. Near-infrared spectroscopy on agricultural harvesters and spectral reflectance of plant canopies have recently been reported as promising components of novel phenotyping platforms. Understanding the genetic basis of complex traits is now within reach with the use of these new techniques.     

Understanding the human condition: experimental strategies in mammalian genetics

Mice have become the mammalian model of choice for the application of genetics in biomedical research due to the evolutionary conservation of physiological systems and their attendant pathologies among all mammals as well as the exceptional power of genetic research technologies in the species. Beginning from aberrant phenotypes, a large number of mouse mutants and natural polymorphisms have been cloned, providing much information about the molecular basis of physiological processes. Additionally, the variable expression of these mutations in different inbred strain backgrounds has demonstrated the importance of modifier genes, which are also susceptible to cloning. Research efforts are keeping pace with these developments. In the area of gene discovery, large, government-funded mutagenesis programs now exist, and as a matter of great practical importance, recent evidence suggests that the same genes may be involved in the natural polymorphisms affecting disease in mice and humans. In parallel, dramatic advances are also being made in our ability to measure physiological processes in mice, and the advent of expression profiling promises revolutionary advances in understanding phenotype at the molecular level. Gene-driven approaches have relied on engineering the mouse genome, including adding, subtracting, and replacing genes and, most recently, the ability to control gene activity reversibly. Together, these multiple advances in our technical abilities have created extraordinary opportunities for future discovery.     

Meeting the meiotic challenge: Specializations in mammalian oocyte spindle formation

Oocytes uniquely accumulate cytoplasmic constituents to support early embryogenesis. This unique specialization is accompanied by acquisition of a large size and by execution of asymmetric meiotic divisions that preserve precious ooplasm through the expulsion of minimal size polar bodies. While often taken for granted, these basic features of oogenesis necessitate unique specializations of the meiotic apparatus. These include a chromatin‐sourced RanGTP gradient that restricts spindle size by defining a spatial domain where meiotic spindles form, acentriolar centrosomes that rely on microtubule organizing centers to form spindle poles, and an actin‐based mechanism for asymmetric spindle positioning. Additionally, localized protein synthesis to support spindle formation is achieved in the spindle forming region, whilst protein synthesis is reduced elsewhere in the ooplasm. This is achieved through enrichment of spindle‐related mRNAs in the spindle forming region combined with local PLK1‐mediated phosphorylation and inactivation of the translational repressor EIF4EBP1. This allows PLK1 to function as an important regulatory nexus through which endogenous and exogenous signals can impact spindle formation and function, and highlights the important role that PLK1 may have in maintaining oocyte quality and fertility.     

Large-scale phenotyping of an accurate genetic mouse model of JNCL identifies novel early pathology outside the central nervous system

Cln3(Δex7/8) mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3(Δex7/8) mice. Homozygous Cln3(Δex7/8) mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10-14 weeks of age. Homozygous Cln3(Δex7/8) mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12-13 week old homozygous Cln3(Δex7/8) mice, which were also seen to a lesser extent in heterozygous Cln3(Δex7/8) mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15-16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3(Δ) (ex7/8) mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3(Δ) (ex7/8) neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3(Δ) (ex7/8) mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3(Δ) (ex7/8) mice that merit further study for JNCL biomarker development.     

PATHBIO: an international training program for precision mouse phenotyping

Mammalian Genome - Design and production of genetically engineered mouse strains by individual research laboratories, research teams, large-scale consortia, and the biopharmaceutical industry have...     

The collaborative cross: a recombinant inbred mouse population for the systems genetic era

The mouse is the most extensively used mammalian model for biomedical and aging research, and an extensive catalogue of laboratory resources is available to support research using mice: classical inbred lines, genetically modified mice (knockouts, transgenics, and humanized mice), selectively bred lines, consomics, congenics, recombinant inbred panels, outbred and heterogeneous stocks, and an expanding set of wild-derived strains. However, these resources were not designed or intended to model the heterogeneous human population or for a systematic analysis of phenotypic effects due to random combinations of uniformly distributed natural variants. The Collaborative Cross (CC) is a large panel of recently established multiparental recombinant inbred mouse lines specifically designed to overcome the limitations of existing mouse genetic resources for analysis of phenotypes caused by combinatorial allele effects. The CC models the complexity of the human genome and supports analyses of common human diseases with complex etiologies originating through interactions between allele combinations and the environment. The CC is the only mammalian resource that has high and uniform genomewide genetic variation effectively randomized across a large, heterogeneous, and infinitely reproducible population. The CC supports data integration across environmental and biological perturbations and across space (different labs) and time.     

Selective phenotyping for increased efficiency in genetic mapping studies

The power of a genetic mapping study depends on the heritability of the trait, the number of individuals included in the analysis, and the genetic dissimilarity among them. In experiments that involve microarrays or other complex physiological assays, phenotyping can be expensive and time-consuming and may impose limits on the sample size. A random selection of individuals may not provide sufficient power to detect linkage until a large sample size is reached. We present an algorithm for selecting a subset of individuals solely on the basis of genotype data that can achieve substantial improvements in sensitivity compared to a random sample of the same size. The selective phenotyping method involves preferentially selecting individuals to maximize their genotypic dissimilarity. Selective phenotyping is most effective when prior knowledge of genetic architecture allows us to focus on specific genetic regions. However, it can also provide modest improvements in efficiency when applied on a whole-genome basis. Importantly, selective phenotyping does not reduce the efficiency of mapping as compared to a random sample in regions that are not considered in the selection process. In contrast to selective genotyping, inferences based solely on a selectively phenotyped population of individuals are representative of the whole population. The substantial improvement introduced by selective phenotyping is particularly useful when phenotyping is difficult or costly and thus limits the sample size in a genetic mapping study.     

Neuroanatomical phenotyping of the mouse brain with three-dimensional autofluorescence imaging

The structural organization of the brain is important for normal brain function and is critical to understand in order to evaluate changes that occur during disease processes. Three-dimensional (3D) imaging of the mouse brain is necessary to appreciate the spatial context of structures within the brain. In addition, the small scale of many brain structures necessitates resolution at the ～10 μm scale. 3D optical imaging techniques, such as optical projection tomography (OPT), have the ability to image intact large specimens (1 cm(3)) with ～5 μm resolution. In this work we assessed the potential of autofluorescence optical imaging methods, and specifically OPT, for phenotyping the mouse brain. We found that both specimen size and fixation methods affected the quality of the OPT image. Based on these findings we developed a specimen preparation method to improve the images. Using this method we assessed the potential of optical imaging for phenotyping. Phenotypic differences between wild-type male and female mice were quantified using computer-automated methods. We found that optical imaging of the endogenous autofluorescence in the mouse brain allows for 3D characterization of neuroanatomy and detailed analysis of brain phenotypes. This will be a powerful tool for understanding mouse models of disease and development and is a technology that fits easily within the workflow of biology and neuroscience labs.     

Sleep phenotyping in a mouse model of extreme trait anxiety

Background

There is accumulating evidence that anxiety impairs sleep. However, due to high sleep variability in anxiety disorders, it has been difficult to state particular changes in sleep parameters caused by anxiety. Sleep profiling in an animal model with extremely high vs. low levels of trait anxiety might serve to further define sleep patterns associated with this psychopathology.

Methodology/Principal Findings

Sleep-wake behavior in mouse lines with high (HAB), low (LAB) and normal (NAB) anxiety-related behaviors was monitored for 24 h during baseline and recovery after 6 h sleep deprivation (SD). The amounts of each vigilance state, sleep architecture, and EEG spectral variations were compared between the mouse lines. In comparison to NAB mice, HAB mice slept more and exhibited consistently increased delta power during non-rapid eye movement (NREM) sleep. Their sleep patterns were characterized by heavy fragmentation, reduced maintenance of wakefulness, and frequent intrusions of rapid eye movement (REM) sleep. In contrast, LAB mice showed a robust sleep-wake rhythm with remarkably prolonged sleep latency and a long, persistent period of wakefulness. In addition, the accumulation of delta power after SD was impaired in the LAB line, as compared to HAB mice.

Conclusions/Significance

Sleep-wake patterns were significantly different between HAB and LAB mice, indicating that the genetic predisposition to extremes in trait anxiety leaves a biological scar on sleep quality. The enhanced sleep demand observed in HAB mice, with a strong drive toward REM sleep, may resemble a unique phenotype reflecting not only elevated anxiety but also a depression-like attribute.     

Measuring the lung function in the mouse: the challenge of size

Measurement of the effects of drugs, mediators and infectious agents on various models of lung disease, as well as assessment of lung function in the intact mouse has the potential for significantly advancing our knowledge of lung disease. However, the small size of the mouse presents significant challenges for the assessment of lung function. Because of compromises made between precision and noninvasiveness, data obtained may have an uncertain bearing on the mechanical response of the lung. Nevertheless, considerable recent progress has been made in developing valid and useful measures of mouse lung function. These advances, resulting in our current ability to measure sophisticated indices of lung function in laboratory animals, are likely to lead to important insights into the mechanisms of lung disease.     

A genetic screen for components of the mammalian RNA interference pathway in Bloom-deficient mouse embryonic stem cells

Genetic screens performed in model organisms have helped identify key components of the RNA interference (RNAi) pathway. Recessive genetic screens have recently become feasible through the use of mouse embryonic stem (ES) cells that are Bloom's syndrome protein (Blm) deficient. Here, we developed and performed a recessive genetic screen to identify components of the mammalian RNAi pathway in Blm-deficient ES cells. Genome-wide mutagenesis using a retroviral gene trap strategy resulted in the isolation of putative homozygous RNAi mutant cells. Candidate clones were confirmed by an independent RNAi-based reporter assay and the causative gene trap integration site was identified using molecular techniques. Our screen identified multiple mutant cell lines of Argonaute 2 (Ago2), a known essential component of the RNAi pathway. This result demonstrates that true RNAi components can be isolated by this screening strategy. Furthermore, Ago2 homozygous mutant ES cells provide a null genetic background to perform mutational analyses of the Ago2 protein. Using genetic rescue, we resolve an important controversy regarding the role of two phenylalanine residues in Ago2 activity.