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 border="0" cellpadding="0" cellspacing="0" style="width:720px;" width="721">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C57BL/6 Mice</td>
			<td style="width:219px;">The Jackson Laboratoy</td>
			<td style="width:119px;">C57BL/6J</td>
			<td style="width:167px;">C57BL/6 mice were used for datasets related to Rodent Research-1 experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BALB/C Mice</td>
			<td style="width:219px;">Taconic</td>
			<td style="width:119px;">BALB</td>
			<td>BALB/C mice were used for datasets related to Rodent Research-3 experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vivarium Cages</td>
			<td style="width:219px;">Charles River Laboratory</td>
			<td style="width:119px;"></td>
			<td>Standard murine cages purchased from Charles River Laboratory</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rodent Habitat</td>
			<td style="width:219px;">NASA</td>
			<td style="width:119px;"></td>
			<td>This cage and all components are built internally at NASA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNAlater</td>
			<td style="width:219px;">ThermoFisher Scientific</td>
			<td style="width:119px;">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 ...