一种确定环境富集对小鼠结肠肿瘤模型结肠菌群生物多样性影响的方法
Summary
环境富集 (EE) 是一种动物住房环境, 用于揭示生活方式、压力和疾病之间联系的机制。该协议描述了一个使用结肠肿瘤发生的小鼠模型和 EE 的过程, 专门定义可能影响动物死亡率的微生物群落生物多样性的变化。
Abstract
最近的几项研究表明生活在丰富环境中对改善人类疾病的有益影响。在小鼠体内, 环境富集通过激活小鼠免疫系统来减少肿瘤的发生, 或者通过刺激伤口修复反应, 包括改善微生物多样性, 在肿瘤微环境中影响到动物生存。这里提供了一个详细的程序, 以评估环境丰富的影响, 微生物的生物多样性的小鼠结肠肿瘤模型。对动物育种的注意事项和动物基因型和小鼠群体整合的考虑进行了描述, 这些都最终影响到微生物的生物多样性。听从这些预防措施可能会使微生物群的传播更加均匀, 从而减轻非治疗依赖的影响, 从而混淆研究结果。此外, 在这个过程中, 微生物群的变化的特点是使用 16S rDNA 测序 DNA 分离从远端结肠收集的长期环境丰富的粪便。肠道菌群失衡与炎症性肠病和结肠癌的发病机制有关, 同时也与肥胖和糖尿病等有关。重要的是, 这项用于 EE 和微生物分析的协议可以用来研究微生物病机在各种疾病中的作用, 在这种情况下, 存在能对人类疾病进行重述的健壮的老鼠模型。
Introduction
环境富集研究利用复杂的房屋参数影响社会刺激 (大型住房笼、大群动物)、认知刺激 (木屋、隧道、筑巢材料、平台) 和体力活动 (跑步车轮)。EE 已被许多实验室利用, 以了解增加的活动和改善社会和认知交互作用的疾病的启动和进展使用了广泛的老鼠模型, 包括理发诱发脱发, 阿尔茨海默病,Rett 综合征, 和一些肿瘤和消化系统疾病模型1,2,3,4,5,6。
为了研究小鼠结肠肿瘤的发生, 开发了几种小鼠模型。也许最明确定义的模型是ApcMin 鼠标。在 1990年7的威廉鸽子实验室中开发了Apc最小鼠标, 并已被用作Apc基因突变的小鼠模型, 通常与人结直肠癌有关。与窝藏apc突变的人类相比, apc最小小鼠主要发育小肠肿瘤, 很少发生结肠肿瘤。但是, Tcf4 使等位基因在Tcf4中具有单一的敲击挖空异型突变, 大大增加了与ApcMin 等位基因8结合的结肠肿瘤发生。最近, 这种小鼠结肠肿瘤发生模型被用来确定 EE 对结肠肿瘤发生的影响6。在 Bice et al中。研究, EE 对四种不同的小鼠的生理和表型效应 (野类型(Tcf4)、Tcf4 多值/+ apc++、 ++ apc Min/++ 、和 Tcf4过度已定义 ApcMin))。也许最有趣的发现是, EE 显著增加了男性和女性结肠肿瘤动物的寿命。这表明, EE 可能会减少至少一些与结肠肿瘤发生有关的症状, 并改善动物的健康。值得注意的是, 这种提高雄性的寿命并不是减少肿瘤发生的直接结果, 而是与引发肿瘤创面愈合反应有关, 包括改良的微生物多样性6。
一些 EE 具体的研究发表了有趣的结果。然而, 从技术角度来看, 重要的结果往往不能翻译给其他实验室。在不同的实验室之间保持相同的 EE 方法是一个令人难以置信的复杂问题, 不仅是由于浓缩设备和使用的住房, 而且还有床上用品、食物、通风、育种、遗传学、室内活动和动物协议。要求, 其中包括9、10、11。其中一个例子是动物整合, 动物必须稳定地融入到老鼠群中, 从而规范遗传背景和饮食成分, 避免非治疗相关的影响。此外, 许多 EE 研究已经完成, 在认识到肠道菌群在疾病的重要性之前, 以及常见的小鼠饲养方法可能影响肠道微生物组的组成的方式10,12。
在 EE 的育种策略和动物安置可以增加压力, 如果不正确地执行。由于 EE 研究利用大量的雄性和雌性动物和多种基因型, 实验性的设置可能是困难的, 因为需要动物从几个凋落物组合。因此, 制定了饲养和断奶的策略, 以允许断奶动物的正确基因型与不同的凋落物相结合。其主要的基本原理是在动物被迁移到实验环境时, 使其在凋落物之间正常化, 并减少压力。微生物群是从大坝中传输的10。为了提供微生物多样性的殖民地, 女性从杰克逊实验室购买, 并纳入该殖民地的一个月前, 实验开始9,10,12。为了进一步规范动物间微生物的生物多样性, 雌性在育种之前是共同居住的。继育种后, 公共住房在饲养和逃生护理幼崽的能力改善了产妇护理的压力水平13,14, 可能进一步促进微生物的规范化。为了防止非 EE 对微生物的影响, 所有实验动物的这一公共住房防止了战斗和额外的压力, 当结合几个雄性从不同的凋落物变成一个实验笼。最后, 所有基因型动物的数量相等, 包括在笼子里。这为改善基因型微生物群落的生物多样性提供了机会, 并消除了 coprophagia (动物的粪便倾向) 或可能的基因型特定行为差异对整体研究的贡献.
该议定书提供了一项战略, 扩大以前的 EE 研究, 包括微生物学研究的已知方面, 包括微生物群的传播和动物群体整合, 以使微生物群规范化, 以便更均匀的微生物种群实验动物之间。注意这些预防措施是必不可少的, 因为非治疗相关的微生物群差异的能力混淆研究结果。消除非 EE 相关的微生物群的变化将使研究人员能够明确界定 EE 在疾病发展和进展过程中对微生物群落组成的作用。
Protocol
这里描述的所有方法都是按照犹他州大学机构动物护理和使用委员会 (IACUC) 批准的协议进行的。 1. 实验设计及电气控制笼安装 注意:为参考, 演示了实验设计的大纲 (图 1)。 设置控件 (NE) 和 EE 保持架 (图 2). 要设置 NE 笼, 请使用缺乏浓缩装置的蒸压常规控制?…
Representative Results
一些研究表明, 精神身体医学的实践改善了健康的结果。同样, 在小鼠, 环境浓缩改善了结果, 包括改善寿命和肿瘤创面修复6。因此, 制定了一个 EE 程序, 目的是确定微生物在这个表型中的作用, 而首先规范化的微生物在实验开始之前 (图 1)。重要的是, 所有繁殖动物在繁殖开始前至少一个月被整合到老鼠群中, 新生幼崽与母亲一起住在…
Discussion
该程序允许分析在正常或肿瘤动物的环境富集后从粪便中分离出来的微生物群。因为这些都是大型实验, 涉及育种, 以获得许多不同性别和基因型的动物, 规范化的动物之间的微生物在实验开始之前, 是必要的, 以避免非 EE 相关的影响微生物多样性.
为了在 NE 和 EE 条件之间保持一致, 繁殖过程是为了确保所有的老鼠最初都接触到相同的微生物, 因此, 预计会有类似的微生物群含量…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢犹他州大学基因组学核心的 Dalley 在图书馆测序, 和 k. 在犹他州大学生物统计学核心提供统计咨询, 并获得国家癌症研究所奖 P30 CA042014 支持的这些技术核心。所述项目得到国家癌症研究所资助 P01 CA073992 和 K01 CA128891 和猎人癌症基金会的支持。
Materials
| Teklad Diets/Harlan Labs Chow | Harlan Labs | 3980X | Standard irradiated chow formulated by Dr. Mario Capecchi in collaboration with Harlan Labs. |
| Cell-Sorb Plus bedding | Fangman Specialties | 82010 | Autoclave prior to use. |
| AIMS Tattooing System For Neonates | AIMS | NEO-9 | 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. |
| Zyfone One Cage 2100 AllerZone Mouse Micro-Isolator System Complete with cage, AllerZone filter top and modular diet delivery system | Lab Products | 82120ZF | Each EE cage requires one of each catalog # 82120ZF, 82100ZF, and 82101ZF, as well as two of 82109ZF. Food is only in one side. |
| Zyfone One Cage 2100 Life Span Enrichment Device | Lab Products | 82109ZF | Each EE cage requires one of each catalog # 82120ZF, 82100ZF, and 82101ZF, as well as two of 82109ZF. Food is only in one side. |
| Zyfone One Cage 2100 Cage 13-7/8" Length X 19-1/16" Width X 7-3/4" Depth | Lab Products | 82100ZF | Each EE cage requires one of each catalog # 82120ZF, 82100ZF, and 82101ZF, as well as two of 82109ZF. Food is only in one side. |
| Zyfone One Cage 2100 AllerZone Micro-Isolator filter top | Lab Products | 82101ZF | Each EE cage requires one of each catalog # 82120ZF, 82100ZF, and 82101ZF, as well as two of 82109ZF. Food is only in one side. |
| Tunnel | Bio-Serv | K3323 or K3332 | Connect cages together and use for enrichment |
| Grommet to connect Tunnel to cages | Fabricated by the University of Utah Machine Shop | n/a | Be certain the material is resistant to chewing and autoclavable |
| Fast-track wheel | Bio-Serv | K3250 or K3251 | Use with mouse igloo and floor |
| Mouse Igloo | Bio-Serv | K3328, K3570 or K3327 | Use with Fast-track wheel and floor |
| Mouse Igloo floor | Bio-Serv | K3244 | Use with mouse Igloo and Fast-Track |
| Mouse Hut | Bio-Serv | K3272, K3102 or K3271 | |
| Crawl Ball | Bio-Serv | K3330 or K3329 | |
| Bio-hut | Bio-Serv | K3352 | Wood pulp hut used for sheltering and nesting |
| Adhesive film | VWR | 60941-072 | Use to temporarily cover drilled hole in large cage to prevent mice from escaping |
| Laminar Flow Ventilated Rack | Techniplast | Bio-C36 | The cabinet we used in this study is not currently supplied. The Bio-C36 is very similar. |
| 1.5 mL Microfuge Tube- RNAse and DNAse free | Any supplier | ||
| QIAamp DNA Stool MiniKit | Qiagen | 51504 | 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 |
| Waterbath (capable of heating to 95) | Any supplier | For 94 degree incubation of stool samples to lyse cells. | |
| Waterbath (capable of heating to 70 degrees) | Any supplier | For 70 degree incubation of stool samples | |
| Ethanol (200 proof) | Sigma Aldrich | E7023 | |
| Fluorometer: Qubit | ThermoFisher Scientific | Q33216 | |
| Qubit dsDNA broad Range Assay Kit | ThermoFisher Scientific | Q32850 | |
| EB Buffer or 10 mM Tris pH 8.5 | Qiagen | 19086 | |
| Experiment specific primers | Any Supplier | ||
| PCR grade water | Any supplier | ||
| 2X KAPA HiFi HotStart Ready Mix | Kapa Biosystems | KK2601 | For Amplicon Amplification (1.25 mL allows 100 rxns). |
| Agarose for running diagnostic gels | Any supplier | ||
| TapeStation High Sensitivity D1000 Screen Tape Trace | Agilent | 5067-5583 | 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. |
| Agencourt AMPure XP Magnetic Beads | Beckman Coulter | A63880 | Magentic beads For PCR cleanup- 5 mL will clean 250 PCR reactions |
| Magnetic stand | Life Technologies | AM10027 | |
| Library Preparation Guide | Illumina | 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. | |
| Unique Dual Indexing | Illumina | Illumina Experiment Manager Software | Freely available at: https://support.illumina.com/sequencing/sequencing_software/experiment_manager/downloads.html |
| Nextera XT 96 Index Kit | Illumina | FC-131-1002 | Used to add barcodes to amplicons |
| MicroAmp Optical 96-well reaction plate | Applied Biosystems/ThermoFisher | N8010560 | |
| TruSeq Index Plate Fixture | Illumina | FC-130-1005 | |
| Adhesive clear plate seal | Applied Biosystems /ThermoFisher | 4360954 | Applied Biosystems/ThermoFisher Microamp adhesive film |
| Sequencing by MiSeq with v3 reagents and dual 300 bp reads | Illumina | MS-102-3003 | |
| PhiX Control Kit | Illumina | FC-110-3001 | |
| Proteinase K (600 mAU/ml) | Qiagen | 19131 | Equivalent to 20 mg/ml of proteinase K. Supplied with QiaAmp kit |
| Data Analysis Tools | Qiime | QIIME software Tools | 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 |
| Step 13.2. | Qiime | FastQ Join method | (http://code.google.com/p/ea-utils ). 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). |
| Step 13.3. | Qiime | De-Novo OTU picking protocol | http://qiime.org/scripts/pick_de_novo_otus.html. |
| Step 13.3.1. | Open Taxonomic Units (OTUs) using Uclust | Edgar, R.C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 26 (19), 2460-2461, doi:10.1093/bioinformatics/btq461 (2010). | |
| Step 13.3.1. | Pynast | Pynast | 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). |
| Step 13.3.1. | Pynast | Pynast_Greengenes | 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 |
| 13.3.1. Note: | Qiime | Multiple Split Libraries | http://qiime.org/scripts/multiple_split_libraries_fastq.html. |
| 13.3.1. Note: | Qiime | Pick de novo OTUs script | http://qiime.org/scripts/pick_de_novo_otus.html |
| Step 13.2.2. | Qiime | Create a mapping file | http://qiime.org/documentation/file_formats.html. |
| Step 13.2.2. | Qiime | Validate a mapping file | http://qiime.org/scripts/validate_mapping_file.html. |
| Step 13.3.3. | Qiime | Link the OTU to sample description to mapping file | http://qiime.org/scripts/make_otu_network.html. |
| Step 13.3.4. | Qiime | Summarize Taxa through plots | http://qiime.org/scripts/summarize_taxa_through_plots.html. |
| Step 13.3.5. | Qiime | Biome Summarize table | http://biom-format.org/documentation/summarizing_biom_tables.html In this study, all samples were rarified to 20,000 OTUs followed by analysis using alpha rarefaction script in QIIME. |
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Cite This Article
Fuller, A. K., Bice, B. D., Venancio, A. R., Crowley, O. M., Staab, A. M., Georges, S. J., Hidalgo, J. R., Warncke, A. V., Angus-Hill, M. L. A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model. J. Vis. Exp. (132), e57182, doi:10.3791/57182 (2018).