Name: a method to define the effects of environmental enrichment on colon microbiome biodiversity in a mouse colon tumor model
Uploaded: 2018-02-28T13:00:00
Description: Watch this Scientific Journal Video about A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model at JoVE.com

We thank B. Dalley in the University of Utah Genomics core for library sequencing, and K. Boucher in the University of Utah Biostatistics core for statistical advice, and access to these technical cores supported by National Cancer Institute award P30 CA042014. The project described was supported by the National Cancer Institute Grants P01 CA073992 and K01 CA128891 and the Huntsman Cancer Foundation.

All methods described here were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Utah.
1. Experimental Design and EE and Control Cage Setup
Note: For reference, an outline of the experimental design is illustrated (Figure 1).
<ol>
	<li>Set up control (NE) and EE cages (Figure 2).</stron...

<ol>
	<li>Bechard, A., Meagher, R., Mason, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+enrichment+reduces+the+likelihood+of+alopecia+in+adult+C57BL/6J+mice.">Environmental enrichment reduces the likelihood of alopecia in adult C57BL/6J mice.</a> Journal of the American Association for Laboratory Animal Science : JAALAS. 50 (2), 171-174 (2011).</li><li>Jankowsky, J. 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Illumina Available from: <a target="_blank" href="https://suport.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf">https://suport.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf</a> (2017)</li><li>Illumina Experiment Manager. Illumina Available from: <a target="_blank" href="https://www.illumina.com/informatics/research/experimental-design/illumina-experiment-manager.html">https://www.illumina.com/informatics/research/experimental-design/illumina-experiment-manager.html</a> (2017)</li><li>Caporaso, J. 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This procedure allows for the analysis of microbiota isolated from stool following environmental enrichment of normal or tumor bearing animals. Because these are large experiments which involve breeding to obtain many animals of different sexes and genotypes, normalizing the microbiome between animals prior to commencement of the experiment is essential to avoid non-EE related effects on microbiome biodiversity.
For consistency between NE and EE conditions, the breeding process is conducted to...

Environmental enrichment (EE) studies utilize complex housing parameters to affect social stimulation (large housing cages, larger groups of animals), cognitive stimulation (huts, tunnels, nesting materials, platforms) and physical activity (running wheels). EE has been utilized by many labs to understand the effects of increased activity and improved social and cognitive interactions on disease initiation and progression using a wide array of mouse models, including barbering induced alopecia, Alzheimer&#39;s disease, Rett syndrome, and several tumor and digestive disease models1,2,3,4,5,6.
Several mouse models have been developed to study colon tumorigenesis in mice. Perhaps the most well-defined model is the ApcMin mouse. The ApcMin&#160;mouse was developed in the laboratory of William Dove in 19907, and has been used as a mouse model of mutations in the APC gene that are commonly associated with human colorectal cancer. In contrast to humans harboring APC mutations, ApcMin mice primarily develop small intestinal tumors, with very rare occurrence of colon tumors. However, a Tcf4Het allele with a single knockin-knockout heterozygous mutation in Tcf4, vastly increases colon tumorigenesis when combined with the ApcMin&#160;allele8. Recently, this mouse model of colon tumorigenesis has been used to determine the effects of EE on colon tumorigenesis6. In the Bice et al. study, the physiological and phenotypic effects of EE on males and females of four different mouse lines (wild-type (WT), Tcf4Het/+ Apc+/+, Tcf4+/+ ApcMin/+, and Tcf4Het/+ApcMin/+)) were defined. Perhaps the most interesting finding was that EE significantly increases the lifespan of both male and female colon tumor-bearing animals. This demonstrated that EE may reduce at least some of the symptoms associated with colon tumorigenesis, and improve animal health. Remarkably, this improved lifespan in males is not a direct result of reduced tumorigenesis, and instead was linked to the initiation of a tumor wound healing response, including improved microbiome biodiversity6.
Several EE specific studies have been published with interesting results. However, from a technical standpoint, important results are often not translatable to other laboratories. Maintaining identical EE methodologies between different laboratories is an incredibly complex issue, not only due to enrichment devices and housing used, but also bedding, food, ventilation, breeding, genetics, activity in the room, and animal protocol requirements, among others9,10,11. One example is animal integration, where animals must be stably integrated into the mouse colony, therefore normalizing genetic background and diet composition, to avoid non-treatment related effects. Further, many EE studies have been completed prior to the realization of the importance of the microbiome in disease, and the way that common mouse husbandry practices can affect the composition of the gut microbiome10,12.
Breeding strategy and animal placement in EE can increase stress if not performed properly. Since EE studies utilize large numbers of both male and female animals and multiple genotypes, experimental setup can be difficult given the requirement for animals from several litters to be combined. Therefore, a breeding and weaning strategy was developed to allow for combining of weaned animals of the correct genotype from different litters. The primary rationale for this was to normalize the microbiota among litters and to reduce stress when animals were moved to the experimental environment. The microbiome was transmitted from the dam10. To provide microbial diversity to the colony, females were purchased from Jackson Labs and integrated into the colony for one month before the experiment began9,10,12. To further normalize microbiome biodiversity between animals, females were co-housed prior to breeding. Following breeding, communal housing during rearing and the ability to escape nursing pups improved the stress levels of maternal care13,14, possibly furthering microbiome normalization. To prevent non-EE related effects on the microbiome, this communal housing of all experimental animals prevented fighting and additional stress that occurred when combining several males from different litters into one experimental cage. Finally, equal numbers of animals of all genotypes were included in the cages. This provided the opportunity for improved microbiota biodiversity across genotypes, and removed the contribution of coprophagia (the animal&#39;s tendency to consume stool) or possible genotype-specific behavioral differences to the overall study.
This protocol provides a strategy that expands previous EE studies to include known aspects of microbiome research, including microbiota transmission and animal colony integration for microbiota normalization, to enable more uniform microbiome populations between experimental animals. Heeding these precautions is essential due to the ability of non-treatment related microbiota differences to confound study findings. Eliminating non-EE related microbiota changes will enable researchers to specifically define the role of EE on microbiota composition during disease development and progression.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Teklad Diets/Harlan Labs Chow</td>
			<td >Harlan Labs</td>
			<td >3980X</td>
			<td >Standard irradiated chow formulated by Dr. Mario Capecchi in collaboration with Harlan Labs.</td>
		</tr>
		<tr >
			<td >Cell-Sorb Plus bedding</td>
			<td >Fangman Specialties</td>
			<td >82010</td>
			<td >Autoclave prior to use.</td>
		</tr>
		<tr >
			<td >AIMS Tattooing System For Neonates</td>
			<td >AIMS</td>
			<td >NEO-9</td>
			<td >https://animalid.com/neonate-rodent-tattoo-identification/32. Other animal grade tattoo systems and inks can be used with similar results including the Aramis Micro Tattoo Kit.</td>
		</tr>
		<tr >
			<td >Zyfone One Cage 2100 AllerZone Mouse Micro-Isolator System Complete with cage, AllerZone filter top and modular diet delivery system</td>
			<td >Lab Products</td>
			<td >82120ZF</td>
			<td >Each EE cage requires one of each catalog # 82120ZF, 82100ZF, and 82101ZF, as well as two of 82109ZF. Food is only in one side.</td>
		</tr>
		<tr >
			<td >Zyfone One Cage 2100 Life Span Enrichment Device</td>
			<td >Lab Products</td>
			<td >82109ZF</td>
			<td >Each EE cage requires one of each catalog # 82120ZF, 82100ZF, and 82101ZF, as well as two of 82109ZF. Food is only in one side.</td>
		</tr>
		<tr >
			<td >Zyfone One Cage 2100 Cage 13-7/8&#34; Length X 19-1/16&#34; Width X 7-3/4&#34; Depth</td>
			<td >Lab Products</td>
			<td >82100ZF</td>
			<td >Each EE cage requires one of each catalog # 82120ZF, 82100ZF, and 82101ZF, as well as two of 82109ZF. Food is only in one side.</td>
		</tr>
		<tr >
			<td >Zyfone One Cage 2100 AllerZone Micro-Isolator filter top</td>
			<td >Lab Products</td>
			<td >82101ZF</td>
			<td >Each EE cage requires one of each catalog # 82120ZF, 82100ZF, and 82101ZF, as well as two of 82109ZF. Food is only in one side.</td>
		</tr>
		<tr >
			<td >Tunnel</td>
			<td >Bio-Serv</td>
			<td >K3323 or K3332</td>
			<td >Connect cages together and use for enrichment</td>
		</tr>
		<tr >
			<td >Grommet to connect Tunnel to cages</td>
			<td >Fabricated by the University of Utah Machine Shop</td>
			<td >n/a</td>
			<td >Be certain the material is resistant to chewing and autoclavable</td>
		</tr>
		<tr >
			<td >Fast-track wheel</td>
			<td >Bio-Serv</td>
			<td >K3250 or K3251</td>
			<td >Use with mouse igloo and floor</td>
		</tr>
		<tr >
			<td >Mouse Igloo</td>
			<td >Bio-Serv</td>
			<td >K3328, K3570 or K3327</td>
			<td >Use with Fast-track wheel and floor</td>
		</tr>
		<tr >
			<td >Mouse Igloo floor</td>
			<td >Bio-Serv</td>
			<td >K3244</td>
			<td >Use with mouse Igloo and Fast-Track</td>
		</tr>
		<tr >
			<td >Mouse Hut</td>
			<td >Bio-Serv</td>
			<td >K3272, K3102 or K3271</td>
			<td ></td>
		</tr>
		<tr >
			<td >Crawl Ball</td>
			<td >Bio-Serv</td>
			<td >K3330 or K3329</td>
			<td ></td>
		</tr>
		<tr >
			<td >Bio-hut</td>
			<td >Bio-Serv</td>
			<td >K3352</td>
			<td >Wood pulp hut used for sheltering and nesting</td>
		</tr>
		<tr >
			<td >Adhesive film&#160;</td>
			<td >VWR</td>
			<td >60941-072</td>
			<td >Use to temporarily cover drilled hole in large cage to prevent mice from escaping</td>
		</tr>
		<tr >
			<td >Laminar Flow Ventilated Rack</td>
			<td >Techniplast</td>
			<td >Bio-C36</td>
			<td >The cabinet we used in this study is not currently supplied. The Bio-C36 is very similar.</td>
		</tr>
		<tr >
			<td >1.5 mL Microfuge Tube- RNAse and DNAse free</td>
			<td >Any supplier</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >QIAamp DNA Stool MiniKit</td>
			<td >Qiagen</td>
			<td >51504</td>
			<td >This kit supplies reagents for 50 DNA preparations. Stool Lysis Buffer=ASL; Guanidinium Chloride Lysis Buffer= AL; Wash Buffer 1 with Guanidinium Chloride= AW1; Wash Buffer 2= AW2; Elution Buffer with EDTA=AE</td>
		</tr>
		<tr >
			<td >Waterbath (capable of heating to 95)</td>
			<td >Any supplier</td>
			<td ></td>
			<td >For 94 degree incubation of stool samples to lyse cells.</td>
		</tr>
		<tr >
			<td >Waterbath (capable of heating to 70 degrees)</td>
			<td >Any supplier</td>
			<td ></td>
			<td >For 70 degree incubation of stool samples&#160;</td>
		</tr>
		<tr >
			<td >Ethanol (200 proof)</td>
			<td >Sigma Aldrich</td>
			<td >E7023</td>
			<td ></td>
		</tr>
		<tr >
			<td >Fluorometer: Qubit</td>
			<td >ThermoFisher Scientific</td>
			<td >Q33216</td>
			<td ></td>
		</tr>
		<tr >
			<td >Qubit dsDNA broad Range Assay Kit</td>
			<td >ThermoFisher Scientific</td>
			<td >Q32850</td>
			<td ></td>
		</tr>
		<tr >
			<td >EB Buffer or 10 mM Tris pH 8.5</td>
			<td >Qiagen</td>
			<td >19086</td>
			<td ></td>
		</tr>
		<tr >
			<td >Experiment specific primers</td>
			<td >Any Supplier</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >PCR grade water</td>
			<td >Any supplier</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >2X KAPA HiFi HotStart Ready Mix&#160;</td>
			<td >Kapa Biosystems</td>
			<td >KK2601</td>
			<td >For Amplicon Amplification (1.25 mL allows 100 rxns).</td>
		</tr>
		<tr >
			<td >Agarose for running diagnostic gels</td>
			<td >Any supplier</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >TapeStation High Sensitivity D1000 Screen Tape Trace</td>
			<td >Agilent</td>
			<td >5067-5583</td>
			<td >TapeStation or Bioanalyzer instruments are common in Institutional Genomics Cores to analyze library quality . Alternatively a Bioanalyzer DNA1000 Chip (Agilent, 5067-1504) can be used.</td>
		</tr>
		<tr >
			<td >Agencourt AMPure XP Magnetic Beads</td>
			<td >Beckman Coulter</td>
			<td >A63880</td>
			<td >Magentic beads For PCR cleanup- 5 mL will clean 250 PCR reactions</td>
		</tr>
		<tr >
			<td >Magnetic stand</td>
			<td >Life Technologies</td>
			<td >AM10027</td>
			<td ></td>
		</tr>
		<tr >
			<td >Library Preparation Guide</td>
			<td >Illumina</td>
			<td ></td>
			<td >Illumina. 16S Metagenomic Sequencing Library Preparation: Preparing 16S ribosomal RNA Gene Amplicons for the Illumina MiSeq System. https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf.</td>
		</tr>
		<tr >
			<td >Unique Dual Indexing</td>
			<td >Illumina</td>
			<td >Illumina Experiment Manager Software</td>
			<td >Freely available at: https://support.illumina.com/sequencing/sequencing_software/experiment_manager/downloads.html</td>
		</tr>
		<tr >
			<td >Nextera XT 96 Index Kit</td>
			<td >Illumina</td>
			<td >FC-131-1002</td>
			<td >Used to add barcodes to amplicons</td>
		</tr>
		<tr >
			<td >MicroAmp Optical 96-well reaction plate</td>
			<td >Applied Biosystems/ThermoFisher</td>
			<td >N8010560</td>
			<td ></td>
		</tr>
		<tr >
			<td >TruSeq Index Plate Fixture</td>
			<td >Illumina</td>
			<td >FC-130-1005</td>
			<td ></td>
		</tr>
		<tr >
			<td >Adhesive clear plate seal</td>
			<td >Applied Biosystems /ThermoFisher</td>
			<td >4360954</td>
			<td >Applied Biosystems/ThermoFisher Microamp adhesive film</td>
		</tr>
		<tr >
			<td >Sequencing by MiSeq with v3 reagents and dual 300 bp reads</td>
			<td >Illumina</td>
			<td >MS-102-3003</td>
			<td ></td>
		</tr>
		<tr >
			<td >PhiX Control Kit</td>
			<td >Illumina</td>
			<td >FC-110-3001</td>
			<td ></td>
		</tr>
		<tr >
			<td >Proteinase K (600 mAU/ml)</td>
			<td >Qiagen</td>
			<td >19131</td>
			<td >Equivalent to 20 mg/ml of proteinase K. Supplied with QiaAmp kit</td>
		</tr>
		<tr >
			<td >Data Analysis Tools</td>
			<td >Qiime</td>
			<td >QIIME software Tools</td>
			<td >Installation may differ based on your system and the QIIME website describes several options (http://qiime.org/install/install.html). For this study, MacQIIME software package 1.9.1 was utilized (compiled by Werner Lab, SUNY, http://www.wernerlab.org/software/macqiime</td>
		</tr>
		<tr >
			<td >Step 13.2.</td>
			<td >Qiime</td>
			<td >FastQ Join method</td>
			<td >&#160;(http://code.google.com/p/ea-utils &#160;).&#160; For this study Multiple join paired ends was used http://qiime.org/scripts/multiple_join_paired_ends.html. Aronesty, E. ea-utils: Command-line tools for processing biological sequencing data. Expression Analysis, Durham, NC. (2011).</td>
		</tr>
		<tr >
			<td >Step 13.3.</td>
			<td >Qiime</td>
			<td >De-Novo OTU picking protocol</td>
			<td >http://qiime.org/scripts/pick_de_novo_otus.html.</td>
		</tr>
		<tr >
			<td >Step 13.3.1.</td>
			<td ></td>
			<td >Open Taxonomic Units (OTUs) using Uclust</td>
			<td >Edgar, R.C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 26 (19), 2460-2461, doi:10.1093/bioinformatics/btq461 (2010).</td>
		</tr>
		<tr >
			<td >Step 13.3.1.</td>
			<td >Pynast</td>
			<td >Pynast</td>
			<td >Caporaso, J.G. et al. PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics. 26 (2), 266-267, doi:10.1093/bioinformatics/btp636 (2010).&#160;</td>
		</tr>
		<tr >
			<td >Step 13.3.1.</td>
			<td >Pynast</td>
			<td >Pynast_Greengenes</td>
			<td >DeSantis, T.Z. et al. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol. 72 (7), 5069-5072, doi:10.1128/AEM.03006-05 (2006). Greengenes version 13_8 was used in this study</td>
		</tr>
		<tr >
			<td >13.3.1. Note:&#160;</td>
			<td >Qiime</td>
			<td >Multiple Split Libraries</td>
			<td>http://qiime.org/scripts/multiple_split_libraries_fastq.html.</td>
		</tr>
		<tr >
			<td >13.3.1. Note:&#160;</td>
			<td >Qiime</td>
			<td >Pick de novo OTUs script</td>
			<td>http://qiime.org/scripts/pick_de_novo_otus.html&#160;</td>
		</tr>
		<tr >
			<td >Step 13.2.2.</td>
			<td >Qiime</td>
			<td >Create a mapping file</td>
			<td>http://qiime.org/documentation/file_formats.html.</td>
		</tr>
		<tr >
			<td >Step 13.2.2.</td>
			<td >Qiime</td>
			<td >Validate a mapping file</td>
			<td>http://qiime.org/scripts/validate_mapping_file.html.</td>
		</tr>
		<tr >
			<td >Step 13.3.3.</td>
			<td >Qiime</td>
			<td >Link the OTU to sample description to mapping file</td>
			<td>http://qiime.org/scripts/make_otu_network.html.</td>
		</tr>
		<tr >
			<td >Step 13.3.4.</td>
			<td >Qiime</td>
			<td >Summarize Taxa through plots</td>
			<td>http://qiime.org/scripts/summarize_taxa_through_plots.html.</td>
		</tr>
		<tr >
			<td >Step 13.3.5.</td>
			<td >Qiime</td>
			<td >Biome Summarize table</td>
			<td >http://biom-format.org/documentation/summarizing_biom_tables.html&#160; In this study, all samples were rarified to 20,000 OTUs followed by analysis using alpha rarefaction script in QIIME.</td>
		</tr>
	</tbody>
</table>

a method to define the effects of environmental enrichment on colon microbiome biodiversity in a mouse colon tumor model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Several studies have demonstrated that the practice of mind-body medicine improves health outcomes. Similarly, in mice, environmental enrichment improves outcomes including improved lifespan and tumor wound repair6. Therefore, an EE procedure was developed with the aim of defining the role of microbiota in this phenotype while first normalizing the microbiome prior to the initiation of the experiment (Figure 1). Importantly, all breedi...

Watch this Scientific Journal Video about A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model at JoVE.com

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

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