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。

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)。
<ol><li>
设置控件 (NE) 和 EE 保持架 (图 2).
	<ol>
<li>要设置 NE 笼, 请使用缺乏浓缩装置的蒸压常规控制笼 (表 1)。</li>
		<li>对于大型网箱, 每笼钻一个孔, 足够大, 足以容纳索环和隧道 (材料表)。</li>
		<li>为了建立幼犬饲养和 EE 笼, 连接两个大型蒸压笼与灭菌索环安全隧道和2消毒平台, 以增加地板空间 (表 1)。对于 ee 实验, 提供消毒运行车轮, 隧道, 圆顶, 木屋, 爬行球, 和筑巢材料在 EE 笼。</li>
		<li>将 EE 和 NE 笼放置在通风机架中, 以提供同等的通风。 注意:两个具有2个平台的大型笼子 (表 1) 允许最多12孕妇每幼崽养育设置15 (请参见材料表、文档中的步骤3.2 和表 2)。</li>
		<li>饲料小鼠ad 随意辐照标准周和蒸压反渗透水。</li>
		<li>为小鼠提供无菌寝具材料。 注意:所有使用动物的空笼子和笼子的操作必须在引擎盖里做, 以防止污染。对于大型网箱的笼式操纵, 用胶膜盖住笼中的孔。</li>
	</ol>
</li>
	<li>为育种准备动物。在交配前2周, 在一个大笼 (表 1) 中, 将 15 2 月大的雌鸟组合在一起。在交配前2周, 分别在2月前 littermate 男性。 注意:育种的雌性数量取决于实验和需要的动物数量。在这项研究中, 15 名女性被利用来获得12个插接的雌性, 这是在 1.1.2 (表 2) 中描述的幼犬饲养设置中允许的最大数目。</li>
	<li>为饲养, 结合了陛下和水坝动物, 1 个男性每2位女性。早上的第一个检查应该是阴道插头, 这可能是动物在夜间交配的视觉信号。 注意:将雄性和雌性结合在一起直到每个雌性都插上插头。阴道插头可以在视觉上识别, 如果它是外部的。<ol>
<li>为检测内化插头, 探头插入阴道开口, 如果阴道插头存在, 探头不易插入 (16; 参见4.3.6 节)。 注意:记录早上一个插头被检测为½天, 因为交配发生在夜间。</li>
		<li>一旦雌性有阴道插头, 转移到一个大的幼犬饲养笼与其他交配雌性 (在步骤1.1.2 没有浓缩装置的设置)。</li>
		<li>取代交配的雌性与新的未交配组安置雌配 , 并继续交配 , 以获得最大数量的凋落物在 7 天的时间。 注意:所有动物在7天之内必须有一个交付日期在彼此之间。</li>
	</ol>
</li>
	<li>允许孕妇在集体住房中分娩, 使所有的凋落物都在一个大的幼犬饲养笼中饲养 (在步骤1.1.2 没有浓缩装置的情况下设置)。<ol>
<li>跟踪幼崽数和生日, 并在7天的年龄开始基因分型。<ol>
<li>纹身幼崽在他们的脚趾在7天的年龄与数字代码, 以确定他们13,17。</li>
			<li>用70% 乙醇清洁脚趾, 轻轻地将含有墨水的微纹身装置插入到与脚趾17平行的皮肤表面 (请参阅材料表)。</li>
			<li>用剪刀从7天的老新生儿身上剪下一小块纸巾, 从尾部小贴士中提取出用于基因分型的组织。</li>
		</ol>
</li>
		<li>将基因组 DNA 从组织中分离出来, 使用常规的方法进行 PCR, 如18所述。</li>
		<li>在14-21 天内将动物与母亲的大笼子分开。 注意:确保年长的幼崽能够养活自己, 幼崽继续看护到足够大的年龄以养活自己。</li>
		<li>21-28 天后, 将雄性和雌性动物分别按基因型分布到 NE 或 EE 环境中, 确保每个保持架的每个基因型的比例相等 (图 2A)。 注意:确保每个 NE 或 EE 笼中允许的动物总数是基于 IACUC 允许的最大数目 (表 2)。NE 笼最多有5只动物 (表 2)。在 ee 网箱中, 为了社会刺激, 不少于 20, 并且有空间限制, 在 ee 笼中不允许超过41只动物 (表 2)。</li>
	</ol>
</li>
</ol>2. 16 周龄时的大便收集
<ol><li>在献祭前1至2天开始大便收集, 并在使用无菌工具解剖的当天, 分别收集粪便。 注意:在收集前1-2 天收集凳子可能有助于避免样本丢失, 因为在收集时没有大便存在的可能性。<ol>
<li>从活着的动物那里收集粪便, 小心地把动物背在干净的笼子上。用无菌钳将粪便收集到无菌离心管中。 注意:动物通常会在固定的时候消除粪便, 这使得快速粪便收集直接进入无菌离心管。如果动物不立即大便时, 固定, 把它放到一个干净的笼子里, 等待动物大便 (通常高达1小时)。</li>
		<li>在献祭日收集凳子。<ol>
<li>为了 euthanizing 动物, 把动物放在一个装有小容器的钟罐子里, 里面有一个棉球浸泡在异氟醚中。一旦停止呼吸被观察 (通常在2分钟以后), 放置动物在它的背部允许结肠解剖。</li>
			<li>将70% 乙醇应用于小鼠腹部。</li>
			<li>在尿道开口前用镊子抬起皮肤, 用剪刀沿腹中线切开直至到达胸腔, 然后从第一切口的底部切开至每条腿。折叠的皮肤和使用剪刀切割通过腹膜壁相同的模式。</li>
			<li>用镊子抓住肛门远端结肠, 从直肠解剖分离远端结肠。用镊子将结肠垂直拉出时, 用剪刀切开肠系膜以释放结肠。</li>
			<li>把盲肠下面的结肠切开, 放在滤纸上。使用镊子抬起结肠管的顶端, 打开流明, 允许一侧打开剪刀插入。纵向切开, 远端到近端, 伸展, 纵向打开结肠。</li>
			<li>使用无菌钳从远端结肠收集粪便到无菌离心管。</li>
		</ol>
</li>
	</ol>
</li>
	<li>将大便放在离心管中-80 ˚C, 直到细菌 DNA 分离的时间。 注意:在献祭日, 除大便外, 还收集其他样本, 如全血、血清、血浆、正常和肿瘤组织, 从结肠和小肠、微粒、脂肪组织、等。在研究中解决已定义的问题。</li>
</ol>3. 从粪便中分离出基因组 DNA
注意:利用商业试剂盒将微生物 DNA 从粪便中分离出粪便病原体检测协议。直接清除样品-80 ˚C 冰箱和储存在干冰, 而权衡。
<ol><li>将多达220毫克的大便转移到含有1.4 毫升室温 (RT) 大便溶解缓冲器的清洁离心管 (请参阅材料表)。</li>
	<li>涡流样本1分钟彻底融汇固体 (图 2B)。将悬浮液加热至95˚C 5 分钟, 溶解所有细菌 (包括革兰氏阳性菌)。</li>
	<li>涡流样品十五年代, 然后离心机在 2万 x g 1 分钟, 以颗粒的粪便固体。将上清液转移到2毫升离心管。在每个样品中加入一个片剂, 吸收 PCR 抑制剂, 涡流直至片剂溶解, 并在 RT 中孵化样品1分钟。</li>
	<li>离心样品在 2万 x g 3 分钟和转移上清到一个新的离心管。离心机在 2万 x g 的3分钟整除 15 ul 蛋白酶 K (20 毫克/毫升的股票) 到一个新的1.5 毫升离心管。吸管 200 ul 的样品进入含有蛋白酶 K 的管。</li>
	<li>添加 200 ul 的胍氯裂解缓冲器到管, 漩涡彻底十五年代 (见材料表) 和孵化的样品在70˚C 10 分钟. 将乙醇 (96-100%) 的 200 ul 添加到管中, 并用涡流混合好。</li>
	<li>将硅基自旋柱放置在2毫升的收集管中, 并将样品应用于该列。关闭盖子和离心机1分钟在 2万 x g。</li>
	<li>将该列转移到新的2毫升收集管, 并将 500 ul 的洗涤缓冲器1添加到柱上, 将该列和离心机盖在 2万 x g 的1分钟内. 将该列转移到新的 2 mL 收集管, 并将 500 ul 的洗涤缓冲器2添加到列中, 关闭盖子和离心机在 2万 x g 为3分钟。</li>
	<li>随着瓶盖关闭, 转移到一个新的2毫升收集管, 和离心机为1分钟, 在 2万 x g, 以消除残留洗涤缓冲。通过将含 EDTA 的洗脱缓冲器的 200 ul 添加到膜上 (参见材料表), 将该列转移到标记为离心管和洗脱样品的1.5 毫升。</li>
	<li>关闭瓶盖, 在 RT 上孵化1分钟. 离心机样品1分钟在 2万 x g. 丢弃该列。</li>
</ol>4. PCR 的 DNA 浓度测定和样品制备
注意:利用荧光计和商业上可用的 dsDNA 荧光法测定每个样品中的基因组 DNA 浓度 (参见材料表)。荧光染料必须特别结合双链 DNA。
<ol><li>准备1:200 稀释每个样品 (每样品的1µL 在199µL dsDNA 大师混合物) 和1:50 稀释标准。使用 dsDNA 设置分析荧光计。 注意:pcr 中大量的 dna 可以抑制, 因此, 使用的 dna 量不得超过 pcr 的最终体积的10%。荧光计能够精确测量样品中的 dna, 因为只有与荧光染料绑定的 dna 才会荧光, 从而消除了污染物对最终计算出的 dna 浓度的可能贡献。这一水平的精确定量是必不可少的下游排序应用。</li>
	<li>准备 PCR 模板稀释到 5 ng/ul 适当的体积10毫米三, pH 8.5, 使每个样品的工作模板库存。</li>
	<li>将样品存放在摄氏-20 摄氏度。</li>
</ol>5. 设计引物到16S 期望的 V 区域
<ol><li>设计底漆, 有选择地放大所需的 V 16S rRNA 区域。</li>
	<li>从核糖体数据库项目19中分析带有探针匹配的引物, 以确定各种门的近似命中率。 注意:对于 V1-V3 区域, 当前的研究使用了已发布的引物彼得·博斯哈德向前20, 它在 V1 区域内的位置8和533个反向21之间绑定, 后者在 V3 区域内的位置533绑定。底漆必须包括用于索引的悬置适配器序列。</li>
	<li>在设计底漆时, 请在每个底漆的 5 ' 端包括适配器序列, 如 16S metagenomics 测序库准备22 (表 3) 所建议的那样。</li>
	<li>用墨盒提纯合成这些大底漆。重建干燥底漆和稀释 PCR 工作存货到1微米在10毫米三, pH 值8.5。</li>
</ol>6. 扩增子 PCR 放大 V 区域 (s) 与悬置适配器序列连接22
<ol><li>设置扩增子 PCR 反应组合,如表4所述。</li>
	<li>使用表 5中的参数, 在板上放置一个粘合剂明确的 pcr 钢板印章, 并运行扩增子 pcr。</li>
	<li>选)运行扩增子 PCR 产品在琼脂糖凝胶或高灵敏度的 DNA 检测, 使定量测量的扩增子大小 (见材料表)。 注意:此项研究的扩增子大小为 550 bp(图 3 A).</li>
</ol>7. 用磁珠进行 PCR 清除22
<ol><li>离心扩增子 PCR 板快速在 1000 x g 1 分钟收集凝结。 注意:pcr 管带可以代替 pcr 板, 以减少污染。丢弃管盖, 永不重复使用。</li>
	<li>涡流磁珠均匀分散, 并添加 20 ul 的磁性珠, 每扩增子 PCR 好, 然后吸管的整个音量上下慢10倍。</li>
	<li>在 RT 上孵育5分钟. 将 PCR 盘放在磁性支架上2分钟, 直到收集到磁性珠子, 清液清晰。除去并丢弃上清。</li>
	<li>用 200 ul 新鲜80% 乙醇洗涤珠, 而 PCR 盘在磁性立场和孵化三十年代在 RT 在磁性立场。小心移除上清。</li>
	<li>再重复一次洗涤。现在, 使用一个细吸管提示, 以消除任何残留的乙醇从水井和允许空气干燥10分钟。</li>
	<li>从磁支架上取出 PCR 板, 并在每个井中添加 52.5 ul 10 毫米三 pH 8.5。吸管上下10次, 悬浮珠, 室温孵育2分钟。</li>
	<li>将 pcr 盘转移到磁性支架上, 收集磁性珠子, 将上清 50 ul 转移到干净的 pcr 板上。将粘合剂明确的 PCR 钢板密封贴在盘子上, 贮存在-20 ˚C, 长达一周。</li>
</ol>8. 用于指数 PCR 的平板方案的制备
注意:要生成 V1-V3 库, 请使用索引工具包执行第二次 PCR (请参见材料表)。默认索引方案用于映射每个示例的唯一双索引组合 (图 3B和23)。
<ol><li>确保每个示例都具有2索引引物 (即、双索引) 的唯一组合。</li>
</ol>9. 执行指数 PCR, 将条形码附加到适配器序列中, 如22所述。
<ol><li>将 2.5 ul 的 PCR amplicons (清洁 amplicons) 转移到一个新的96井板, 并放置在一个指数板夹具中, 以协助编制索引。</li>
	<li>将索引1和索引2引物排列为在预准备的板图形 (图 3B) 的示例中。 注意:提供视觉提示, 以避免底漆混合: 索引2底漆管应该有白帽和明确的解决方案, 而指数1底漆管应该有橙色帽和黄色溶液。</li>
	<li>按照表 6中的说明, 装配索引 PCR 混合反应。混合吹打上下10次。在室温下以 1000 x g 为1分钟, 用粘合剂透明的 PCR 钢板密封和离心机盖住。</li>
	<li>使用表 7中的参数运行索引 PCR。</li>
</ol>10. 纯化最终 PCR 库
注意:这种 pcr 清理是相同的步骤7以上, 并使用磁性珠进行 pcr 清除指数 pcr22。
<ol><li>将 PCR 板从步骤10快速的离心到 1000 x g 以1分钟收集凝结。</li>
	<li>将磁珠均匀地分散, 然后将20的磁珠 ul 添加到每个扩增子 PCR 井中, 然后将整个音量上下缓慢10次混合。</li>
	<li>在 RT 孵育5分钟。</li>
	<li>将 PCR 盘放在磁性支架上2分钟, 直到收集磁性珠子, 清液清晰。除去并丢弃上清。</li>
	<li>用 200 ul 新鲜80% 乙醇洗涤珠, 而 PCR 盘在磁性立场和三十年代在室温下孵化在磁性立场。小心移除上清。</li>
	<li>再重复一次洗涤。</li>
	<li>第二次清洗后, 使用细吸管尖端, 从油井中除去任何残留的乙醇, 并允许空气干燥10分钟。</li>
	<li>从磁支架上取出 PCR 板, 并在每个井中添加 52.5 ul 10 毫米三 pH 8.5。吸管上下10次, 悬浮珠, 室温孵育2分钟。</li>
	<li>将 pcr 盘转移到磁性支架上, 收集磁性珠子, 将上清 50 ul 转移到干净的 pcr 板上。将粘合剂明确的 PCR 钢板密封贴在盘子上, 贮存在-20 ˚C, 长达一周。</li>
	<li>选)在琼脂糖凝胶或高灵敏度的 DNA 检测上运行指数 PCR 产品, 使定量测量扩增子大小 (参见材料表)。 注意:此项研究的最终索引库大小为 668 bp (图 3C D)。</li>
</ol>11. 量化、规范化和汇集索引库以进行排序
<ol><li>用荧光计和 dsDNA 荧光检测试剂盒确定每个样品的 DNA 浓度 (见材料表)。<ol>
<li>准备1:200 稀释样品 (每个样品的1µL 在199µL dsDNA 主要混合物, 包括缓冲和试剂) 为每个索引的样品和标准 (190 µL dsDNA 大师混合物和10µL 标准)。使用 dsDNA 设置分析荧光计。</li>
	</ol>
</li>
	<li>在 DNA 浓度计算之后, 通过计算平均库大小来规范化库。通过对引物的适配器长度、索引长度和 V 扩增子大小进行求和, 并通过琼脂糖凝胶查看产品, 确定实际大小与计算大小类似(图 3 C, 请参见22)。<ol>
<li>或者, 利用高灵敏度的 dna 化验, 可以定量测量 dna 完整性、扩增子大小和浓度 (材料表,图 3 D). 注意:在本研究中, 根据引物长度、指数长度和 V1-V3 扩增子大小计算了平均库大小。平均大小是 668 bp。</li>
		<li>使用表 8中的公式对样本集中进行规范化。</li>
	</ol>
</li>
	<li>稀释样品到4毫微米和水池 5 ul 从每4毫微米样品入一根管子为测序。</li>
</ol>12. 使用下一代测序系统对库进行排序并分析数据
<ol><li>对库进行排序。 注意:在这项研究中, 犹他大学的高通量基因组学核心执行了库变性和样本排序 (如6,22中所述)。</li>
	<li>分析数据。 注意:若要将数据与池中的示例分开, 则标识并分隔索引读取 (如22所述)。</li>
	<li>生成 FASTQ 文件并将其用于后续数据分析。</li>
</ol>13. 分析16S 扩增子库中的测序数据
注意:此步骤按照 Bice et al中的说明执行, 20176。
<ol><li>安装可自由使用的数据分析工具 (请参阅材料表;24)。</li>
	<li>从序列数据中装配 demultiplexed fastq 文件 (如25、26中所述)。丢弃所有未组装序列。</li>
	<li>在从头开始打开分类单元 (OTU) 领料协议后执行分析 (如27中所述)。<ol>
<li>Bin 序列放入单个 fastq 文件中, sampleID 和组序列具有97% 或更大的相似性 OTUs, 如28所述。将核心集的代表性序列与最小序列长度为150和 75%% 的标识29,30对齐。按描述的28分配分类。 注意:可以作废和分析示例31, 其次是分类分配和 OTU 表构造32。</li>
		<li>创建一个映射文件, 用于标识要链接到示例标识的示例的描述性名称和特征, 并验证映射文件33,34。</li>
		<li>使用映射文件35建立一个将 OTUs 链接到示例描述的 OTU 网络。</li>
		<li>通过通过地块36汇总分类, 计算分类摘要的相对丰度。</li>
		<li>根据样品的均匀测序深度, 探索样品的α多样性。要定义 alpha 多样性的适当深度, 请使用 "生物群落汇总表" 命令汇总每个示例中观察到的总计数, 如37所述。</li>
	</ol>
</li>
</ol>

<ol>
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作者声明他们没有利益冲突。

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...

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。

<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

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。

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...

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

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。

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

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

a-method-to-define-effects-environmental-enrichment-on-colon

Research

JoVE Journal

Cancer Research

8.7K 次观看.  University of Utah. 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。

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

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

科研

癌症研究

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

结肠-26癌荷瘤小鼠为癌症恶病质的研究模型

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

人地幔细胞淋巴瘤移植小鼠模型中肿瘤植入的研究

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

骨肉瘤前小鼠模型对肿瘤与间充质干细胞细胞外囊泡介导通信的定义

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

用基因编码荧光传感器评估毒理学氧化应激的成像方法

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

自体胰腺癌小鼠模型研究不可逆电穿孔的影响

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

淋巴结慢病毒北疆-转基因 Organoids 培养的大鼠结肠肿瘤的高灵敏度检测

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

患者衍生的人类泌尿细胞癌和结肠直肠癌肿瘤生长和自发转移的正交异种移植模型

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

人类结肠癌细胞DNA损伤与修复福西的免疫荧光成像

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...