We would like to thank Alison French at NASA Ames Life Science Data Archive for her assistance with obtaining the video related to the Rodent Habitats and overall help with obtaining cage related information. We also like to thank Marla Smithwick at NASA Ames Research Center for her help with obtaining the proper information. Research funding was provided by the GeneLab Project at NASA Ames Research Center, through NASA's Space Biology Program in the Division of Space Life and Physical Sciences Research and Applications (SLPSRA). Any use of trade names is for descriptive purposes only and does not imply endorsement by the US Government.

The authors have nothing to disclose.

The NASA GeneLab platform is a comprehensive omics database and analysis platform that will allow the scientific community to generate novel hypotheses related to space biology. Here we have presented a comprehensive procedure for rodent experiments from the beginning of spaceflight to the generation of novel hypothesis from analyzing data utilizing a publicly available space biology platform. In addition, we have also provided an extensive protocol on an unbiased systems biology analysis for identifying key genes driving the system being studied. We have used our recent study36 as an example of how this protocol is effectively utilized to generate a novel hypothesis for space biology. We hope that this helps investigators better understand how spaceflight experiments are performed and how data from them lead to the data available on GeneLab, and ultimately allow for clearer interpretation of publicly available space biology omics data.
There are several critical steps within our protocol regarding both rodent spaceflight experiments and analysis of the data produced. Understanding the Rodent Habitat setup is critical to develop and design the optimal experiment for spaceflight. This would specifically entail the protocol and description we have provided in step 1 of our protocol. Once an investigator fully understands the different conditions existing in the Rodent Habitats compared to vivarium cages, the biological results being interpreted can be correlated properly to the environmental conditions in space. In additions, modifications to the Rodent Habitat cannot be done, since the Rodent Habitat has been optimally designed and approved by NASA for use of spaceflight.
To interpret the biological results, we have provided a thorough protocol on every step involved from uploading your data to GeneLab to analysis of the data to generate novel space biology hypothesis. Although all the steps are important in understanding how to generate data, the most critical steps for data analysis are steps 9 and 10. Step 9 provides a protocol to analyze transcriptomic data using an unbiased systems biology method to determine genes/pathways that are truly driving the experimental condition being analyzed. Step 10 is critical as it provides users with an easy methodology to analyze omics GeneLab datasets using the GeneLab platform. Modifications to the protocol provided can be done for some steps regarding analyzing data. Specifically, steps 9.4&#8211;9.6 can be done using R programming or any other favorite tools that the user prefers. Depending on the dataset, different statistics and fold-change cutoffs can be used to determine the significantly regulated genes. In addition, for determining the key genes in steps 9.5 and 9.6, the user can modify this protocol and use any tool that utilizes the significantly regulated genes to predict functions. The important concept is that using multiple predictive functional omics tools allows for determination of genes involved with the majority of functions being regulated in the system being studied.
The GeneLab platform continues to develop, and while the analyses described here were performed after data download, the next phase of GeneLab will allow for analysis of omics data directly on GeneLab platform, which will provide an easy workflow to generate processed data for higher-order analysis. Moreover, whereas we have focused on a protocol for interpreting transcriptomic data, GeneLab contains a wide variety of omics data including proteomic, genomic, metabolomic, and epigenomic data. The eventual platform will contain pipelines and guidelines for analysis of these different types of omics. The last phase of GeneLab will also implement a system level visualization interface to allow the basic user to easily generate space biology hypotheses.
Lastly, our systems biology analysis provides a unique and unbiased method to determine the key driving genes/pathways in any system being studied using omics datasets. We have used this methodology in several different independent studies with great success to determine the key drivers involved36,45,46,47,48,49,50. In a cancer related omics study, using this methodology we experimentally validated that our predicted key genes/pathways were actually driving the drug treatment response by knocking out the key genes in vitro45. We observed, as we had predicted through this protocol, that the treatment was not effective anymore due to the absence of the key genes. We believe that this unbiased systems biology protocol can be a useful tool to determine key pathways for any omics study.
This protocol provides a quick and efficient method for the generation of novel space biology hypotheses. The data generated from GeneLab can be leveraged by investigators for future funding opportunities, experimental validation, and potential targets for development of countermeasures against microgravity and space radiation. The protocol provided here will permit for future space biology investigations to occur with optimal efficiency to allow for safe long-term space missions.

The overall goal of this manuscript is to provide a clear methodology of how to use NASA&#39;s GeneLab platform1 and how rodent experiments done in space are translated to omics data for analysis. Spacefaring humans are exposed to numerous health risks from altered gravity fields, space radiation, isolation from Earth, and other hostile environmental factors2,3,4,5,6. Biological experiments performed in space and on the ground have helped to define and quantify these risks7,8,9,10,11,12,13,14. In space, these experiments have been conducted on the International Space Station (ISS), the Space Shuttle, and other orbital platforms. Conducting these experiments requires specialized hardware and methodology given the unique concerns of performing experiments in space including limited crew time and the microgravity environment. Various platforms now exist for performing sophisticated experiments in space using plant, animal, and microbial models15.
Rodent models have been particularly important to advancing our understanding of how mammals, including humans, respond to spaceflight. These include the impact of spaceflight on the muscle structure16,17,18 and immune functions19,20,21. The standard vivarium cages used for housing rodents on Earth are not suitable for spaceflight experiments22,23. Therefore, over the years mice and rats have been flown and housed in various cages including the Japanese Aerospace Exploration Agency (JAXA) Habitat Cage24, animal carrying space capsules used on the BION-M1 unmanned Russian satellite25,26,27, the Mice Drawer System (MDS) designed by the Italian Space Agency28,29,30, the NASA Animal Enclosure Module (AEM), and now the NASA Rodent Transporter and Habitats23. Rodent experiments first started on board the Space Shuttle using cages referred to as the Animal Enclosure Module (AEM). This hardware was used in 27 rodent experiments on the Space Shuttle23. The AEM was originally developed for relatively short experiments on-board the shuttle (&#60; 20 days). Since the development of the ISS, the AEMs have been modified for longer duration experiments and are now referred to as Rodent Habitats22,23. The new Rodent Habitats are designed to support long-duration missions in the ISS using the EXpedite the PRocessing of Experiments for Space Station (EXPRESS) Rack interface. Rodent Habitats are substantially different from standard vivarium cages in their dimensions, air flow, filter and exhaust system, and access to food and water (Figure 1). Nevertheless, this hardware has proven to be an effective research platform, enabling key insights into the spaceflight-induced changes to mammalian physiology19,31,32,33,34,35,36.
Large volumes of omics data can now be generated from biological spaceflight experiments including those performed with rodents. Recently, data from these omics experiments have been made publicly available through the NASA GeneLab platform1 which is a comprehensive data repository and analysis platform that allows anyone to develop hypotheses from spaceflight experiments. GeneLab provides tools for the discovery, access, sharing and analysis of data. We utilized GeneLab datasets to show that differences between the standard vivarium cages and specialized Rodent Habitats used in space cause massive differences in the transcriptome of mice36. We analyzed four different publicly available datasets, comparing different tissues from rodents housed in either the Rodent Habitats or standard vivarium cages. Using an unbiased systems biology analysis, we determined that the main drivers and pathways that were changed were consistent with a hypoxic response due to the high CO2 levels caused by higher CO2 concentrations on ISS, which leads to higher CO2 concentrations in the Rodent Habitat given that they are passive systems that take in the ambient air. This demonstrates how scientists can use open source tools and data to generate novel findings with implication on how the environment of the ISS impacts astronaut health.
Here we describe how rodent experiments are performed in space and how data from these experiments can be accessed through an open-source, omic platform related to space biology. We discuss the configuration of the Rodent Habitats used for space missions, and how spaceflight tissues are processed. We also describe how spaceflight omics data can be discovered and accessed on GeneLab and how key factors driving the overall response to spaceflight can be identified36. The specific example we will present on how this protocol is implemented will be comparing the biological differences occurring in rodents housed in Rodent Habitat and the vivarium controls that were published by Beheshti et al.36. It is important to note that ground controls are essential for spaceflight rodent experiments. As described in this protocol, these controls are done with both identical conditions (i.e., CO2 conditions, humidity, temperature, cage dimensions, etc.) in the Rodent Habitats on the ISS and in standard vivarium cages that have the standard environmental (i.e., CO2 conditions, humidity, and temperature) conditions on Earth. The rodents housed in the Rodent Habitat ground controls allow for the direct comparison to rodents in space. While rodents housed in vivarium cages allow for the biological comparison between the different housing (e.g., vivarium cages vs. Rodent hardware). The Rodent Habitat is different than vivarium cages in that it has constant air flow (0.1&#8211;0.3 m/s), a long duration, and a secondary exhaust filter that captures and absorbs the animal waste guided to the exhaust filter by continuous air flow in microgravity. In addition, Rodent Habitats have passive systems and intake ambient air; therefore, they also have higher CO2 concentrations due to elevated levels in the ISS cabin (~5,000 ppm).

<table><tbody><tr><td>C57BL/6 Mice</td><td>The Jackson Laboratoy</td><td>C57BL/6J</td><td>C57BL/6 mice were used for datasets related to Rodent Research-1 experiments</td></tr><tr><td>BALB/C Mice</td><td>Taconic</td><td>BALB</td><td>BALB/C mice were used for datasets related to Rodent Research-3 experiments</td></tr><tr><td>Vivarium Cages</td><td>Charles River Laboratory</td><td></td><td>Standard murine cages purchased from Charles River Laboratory</td></tr><tr><td>Rodent Habitat</td><td>NASA</td><td></td><td>This cage and all components are built internally at NASA</td></tr><tr><td>RNAlater</td><td>ThermoFisher Scientific</td><td>AM7020</td><td>RNAlater is used to store the tissue for further RNA isolation</td></tr></tbody></table>

exploring the effects of spaceflight on mouse physiology using the open access nasa genelab platform

Performing biological experiments in space requires special accommodations and procedures to ensure that these investigations are performed effectively and efficiently. Moreover, given the infrequency of these experiments it is imperative that their impacts be maximized. The rapid advancement of omics technologies offers an opportunity to dramatically increase the volume of data produced from precious spaceflight specimens. To capitalize on this, NASA has developed the GeneLab platform to provide unrestricted access to spaceflight omics data and encourage its widespread analysis. Rodents (both rats and mice) are common model organisms used by scientists to investigate space-related biological impacts. The enclosure that house rodents during spaceflight are called Rodent Habitats (formerly Animal Enclosure Modules), and are substantially different from standard vivarium cages in their dimensions, air flow, and access to water and food. In addition, due to environmental and atmospheric conditions on the International Space Station (ISS), animals are exposed to a higher CO2 concentration. We recently reported that mice in the Rodent Habitats experience large changes in their transcriptome irrespective of whether animals were on the ground or in space. Furthermore, these changes were consistent with a hypoxic response, potentially driven by higher CO2 concentrations. Here we describe how a typical rodent experiment is performed in space, how omics data from these experiments can be accessed through the GeneLab platform, and how to identify key factors in this data. Using this process, any individual can make critical discoveries that could change the design of future space missions and activities.

Determining key drivers from spaceflight transcriptomic data will assist NASA with determining health risks and developing potential countermeasures to combat negative effects on astronaut health. In our recent publication, we have followed the steps above and utilized GeneLab datasets to successfully show a novel finding that CO2 concentrations on the ISS can impact health36. We have also used the technique above in other studies to successfully determine the key factors driving the system being studied45,46,47,48,49,50. Here we will show how the results from using this protocol can be successfully used to determine the key drivers.
In this study, we primarily focused on the biological differences that occur in rodents housed in the Rodent Habits ground controls and the vivarium controls. As described above, it is the key to better understanding these two habitats, which will provide us information on possible confounding factors that can impact health due to the environment on the ISS. For all rodent spaceflight experiments, these ground controls are also essential to determine which biological factors are associated directly with spaceflight or due to the environmental conditions on the ISS. As stated in the protocol, the environmental condition for the vivarium habitat is not exposed to the higher CO2 level that is present for the Rodent Habitat. The vivarium habitat has the normal CO2 level that is present on Earth (currently being 300 to 380 ppm). The temperature and humidity for both habitats are similar.
We used the following datasets from the GeneLab platform to determine the key genes between the rodents housed in the Rodent Habitat ground controls and vivarium ground controls that are responsible for driving the differences between the two habitats: GLDS-21, GLDS-111, GLDS-25, and GLDS-63. Analysis to determine the significant genes was carried out as described above between the Rodent Habitat (previously AEM) and vivarium controls independently for each dataset. PCA plots showed grouping of the biological replicates (Figure 4 shows the PCA plots for GLDS-21). From the pre-processed data, we determined the leading-edge genes from the different GSEA gene sets. Using the genes with 1.2-fold-change (log2), we were able to predict the genes involved with predictions for upstream regulators, canonical pathways, and biofunctions. For each dataset we then found the common/overlapping genes involved for all the genes (Figure 5). These genes are now believed to be driving the response between the rodents in the Rodent Habitats (or AEM) and vivarium controls. Network representation of how these key genes connect shows the central hubs for each dataset being analyzed (Figure 6). For example, MAPK1 is the central hub for STS-108 skeletal muscle tissues from mice (Figure 6A). This would be interpreted as the gene that is driving the key genes and most likely the central player for causing biological differences for mice housed in Rodent Habitats versus the vivarium cages. In our previous work, we discuss how these key genes are associated with CO2 response from the existing scientific literature and how these genes can be responsible for biological changes observed in the mice36.
Taking a systems biology approach, we next determined a &#34;master regulator&#34; that connects all the datasets/tissues and is potentially responsible for universal biological effects in rodents housed in AEMs compared to vivarium cages. This was done by determining the gene from all the datasets that is the most connected when constructing a network from all the key genes. We were able to show that MAPK1 is the most connected gene and central hub from all the key genes (Figure 7). To confirm if MAPK1 might be responsible for biological changes in mice from the higher CO2 levels in AEMs, we looked through the scientific literature for supporting evidence. We found several studies indicating the correlation of MAPK1 with CO259 and hypoxia19,60,61.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58447/58447fig1.jpg" /> 
Figure 1: The Rodent Habitat (previously AEM) compared to the vivarium cages.&#160;(A) Image of the AEM cage provided by NASA (Credits: NASA/Dominic Hart). (B) The standard vivarium cage that is currently used (photo taken by our laboratory). This figure has been modified from Beheshti et al.36. <a href="https://www.jove.com/files/ftp_upload/58447/58447fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58447/58447fig2.jpg" /> 
Figure 2: The Rodent Habitat Hardware System with the three different modules involved during transportation to and from the space missions. The left module (A) is the Rodent Habitat module (previously AEM), the center module (B) is the Transporter, and the right module (C) is the Animal Access Unit (AAU). (D)The&#160;Mouse Transfer Box (MTB). (Credits: NASA/Dominic Hart). <a href="https://www.jove.com/files/ftp_upload/58447/58447fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58447/58447fig3.jpg" /> 
Figure 3:&#160;Example analysis workflow which can be used in the GeneLab Galaxy interface to process RNA-seq data. <a href="https://www.jove.com/files/ftp_upload/58447/58447fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58447/58447fig4.jpg" /> 
Figure 4: Principal component analysis (PCA) of representative dataset after pre-processing steps. GLDS-21 dataset for AEM vs. vivarium cage is shown for the murine skeletal muscle from the STS-118 mission. <a href="https://www.jove.com/files/ftp_upload/58447/58447fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58447/58447fig5.jpg" /> 
Figure 5:&#160;Venn diagram representing what key genes are determined using different pathway prediction tools. <a href="https://www.jove.com/files/ftp_upload/58447/58447fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58447/58447fig6.jpg" /> 
Figure 6: The key genes determined for all conditions and murine tissues between the AEM vs. vivarium cages.&#160;(A-E) Network representation of the key genes for each dataset/rodent tissue. Log2 fold-changes (with a cutoff of 1.2-fold-change) to the gene expression were used to obtain different shades of green for fold-change in downregulated genes, while different shades of red depict fold-change in upregulated genes. The darker the shade of green or red, the greater the fold-change. This figure has been modified from Beheshti et al.36. <a href="https://www.jove.com/files/ftp_upload/58447/58447fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58447/58447fig7.jpg" /> 
Figure 7: Determining the &#34;master regulator&#34; for rodents in Rodent Habitat housing compared to vivarium cages. Connections between all individual key genes (Figure 6) were determined and displayed as a network through IPA. Network is represented as a radial plot with the most connected key gene, MAPK1, in the center. <a href="https://www.jove.com/files/ftp_upload/58447/58447fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure 1: GeneLab-GenomeSpace Integration with ISACreator for Streamlining Data Processing Operations.&#160;&#160;<a href="https://www.jove.com/files/ftp_upload/58447/Supplemental_Figure_1.tif" target="_blank">Please click here to download this figure.</a>
Supplemental Figure 2: Screenshot of GeneLab searches using federation/integration with heterogeneous bioinformatics external databases (GEO, PRIDE, MG-RAST).&#160;<a href="https://www.jove.com/files/ftp_upload/58447/Supplemental_Figure_2.tif" target="_blank">Please click here to download this figure</a>
Supplemental Figure 3: Screenshot of the GeneLab collaborative workspace showing the user account management, and access controls (e.g., private, shared, public folders).&#160;<a href="https://www.jove.com/files/ftp_upload/58447/Supplemental_Figure_3.tif" target="_blank">Please click here to download this figure</a>

Watch this Scientific Journal Video about Exploring the Effects of Spaceflight on Mouse Physiology using the Open Access NASA GeneLab Platform at JoVE.com

Exploring the Effects of Spaceflight on Mouse Physiology using the Open Access NASA GeneLab Platform

The NASA GeneLab platform provides unfettered access to precious omics data from biological spaceflight experiments. We describe how a typical mouse experiment is conducted in space and how data from such experiments can be accessed and analyzed.

Performing biological experiments in space requires special accommodations and procedures to ensure that these investigations are performed effectively ...

exploring-effects-spaceflight-on-mouse-physiology-using-open-access

Research

JoVE Journal

Genetics

12.2K Views.  NASA Ames Research Center. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in de ruimte biologie veld over het genereren van hypothesen om toekomstig onderzoek te helpen met behulp van openbaar beschikbare alle mix gegevens. Het belangrijkste voordeel van deze techniek is dat het kan worden gebruikt als een instrument voor het genereren van ruimtebiologie gegevens. De openbaar beschikbare gegevens over GeneLab maken onderzoeken mogelijk om klinische theorieën te ontwikkelen over ziektetherapieën o...

Video: Exploring the Effects of Spaceflight on Mouse Physiology using the Open Access NASA GeneLab Platform

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

Developmental Biology

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

Behavior

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...

Medicine

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

Biology

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

An Introduction to the Laboratory Mouse: Mus musculus

Mice (Mus musculus) are an important research tool for modeling human disease progression and development in the lab. Despite differences in their size and appearance, mice share a distinct genetic similarity to humans, and their ability to reproduce and mature quickly make them efficient and economical candidate mammals for scientific study.
This video provides a brief overview of mice, both as organisms and in terms of their many advantages as experimental models. The discussion features an introduction to common laboratory mouse strains, including the nude mouse, whose genetic makeup renders them both hairless and immunodeficient. A brief history of mouse research is also offered, ranging from their first use in genetics experiments to Nobel prize-winning discoveries in immunology and neurobiology. Finally, representative examples of the diverse types of research that can be performed in mice are presented, such as classic behavioral tests like the Morris water maze and in-depth investigations of mammalian embryonic development.

Mus musculus Laboratory Mouse as a Model Organism

Mice (Mus musculus) are an important research tool for modeling human disease progression and development in the lab. Despite differences in their size ...

Education

JoVE Science Education

Basic Biology

Biology II: Mouse, Zebrafish, and Chick

Development and Reproduction of the Laboratory Mouse

Successful breeding of the laboratory mouse (Mus musculus) is critical to the establishment and maintenance of a productive animal colony. Additionally, mouse embryos are frequently studied to answer questions about developmental processes. A wide variety of genetic tools now exist for regulating gene expression during mouse embryonic and postnatal development, which can help scientists to understand more about heritable diseases affecting human development.
This video provides an introduction to the reproduction and development of mice. In addition to clarifying the terminology used to describe developmental progression, the presentation reviews key stages of the mouse life cycle. First, major development events that take place in utero are described, with special attention given to the unique layout of early rodent embryos. Next, husbandry protocols are provided for postnatal mice, or pups, including the process of weaning, or removal of pups from their mother&#39;s cage. Since males and females must be separated at this stage to prevent unscheduled mating, the demonstration also reveals how to determine mouse sex. Subsequently, instructions are given for carrying out controlled mouse breeding, including screening for the copulatory plug, which is useful for precisely timed embryonic development. Finally, the video highlights strategies used to investigate the complex processes that govern mouse development, including the generation of genetically altered &#8220;knockout&#8221; mice.

Reproduction and Development of the Laboratory Mouse

Successful breeding of the laboratory mouse (Mus musculus) is critical to the establishment and maintenance of a productive animal colony. Additionally, ...

Exploring the Effects of Spaceflight on Mouse Physiology using the Open Access NASA GeneLab Platform

Samenvatting

Meer video's verkennen