这项工作得到了美国哈特福德大学艺术与科学学院院长办公室的师资研究补助金的支持。

在这项研究中, 动物护理和治疗符合国家卫生机构的政策, 是按照机构的指导方针, 并获得了哈特福德大学动物机构动物保育和使用委员会的批准。
1. 动物的夹带
注: 研究中使用二十周大的雄性 B6129SF2/J 小鼠。
<ol><li>实验前2周, Entrain 小鼠到 12 h 光 (标准架空白光)/12 h 黑暗周期。 注: 这里授时时间 (ZT) 0 对应于灯和 ZT12 关灯, 同时保持所有其他环境因素 (例如,食物、水和室温) 恒定21。</li>
</ol>2. 编写文书、培养基和挑战媒介
<ol><li>高压釜钳和解剖剪刀。裹在铝箔和高压釜的磨砂显微镜幻灯片。</li>
	<li>通过将胎牛血清 (血清) 添加到 RPMI 1640 到最后浓度 10%, 制备培养基。在50毫升管中加入以下 PAMPs, 以培养培养基 (RPMI 10%), 制备10毫升的挑战培养基;LPS (5 µg/毫升), ODN1826 (5 µg/毫升), 热杀死李斯特菌 (108 HKLM/毫升), 或另一玉兰在其建议浓度。</li>
	<li>暖培养基和挑战培养基到37°c 在水浴, 并保持约70毫升不育磷酸盐缓冲盐水 (PBS, pH 7.2) 在室温下。</li>
	<li>添加10毫升培养基, 使用10毫升吸管和吸管帮助50毫升管和放置在冰上。</li>
	<li>将大约30毫升70% 乙醇添加到100毫升烧杯中, 将解剖剪刀和镊子的两端放入烧杯中, 以防止微生物污染。</li>
	<li>在50毫升管中加入10µL β巯基乙醇 (在通风罩下), 为每1毫升的缓冲 RLT 制备 RNA 分离的裂解缓冲液。只制作需要的量 (每个样本600µL, 由大约 1 x 106个单元格组成)。 注意: 缓冲 RLT 是 rna 提取套件的一个组成部分, 它支持 rna 与二氧化硅膜的结合。</li>
</ol>3. 脾隔离和挑战
<ol><li>弄死小鼠在特定的授时时间通过麻醉保持他们在他们的原始笼子和增加 co2到笼子在流动率3升/分钟. 在呼吸停止后继续供应 co2为1分钟。</li>
	<li>通过将拇指和食指放在头骨底部的颈部两侧, 确认颈部脱位死亡。或者, 在头骨底部按一根棒, 同时快速拉 (用另一只手) 尾部或后肢的底部, 使颈椎从头骨22分离。</li>
	<li>用70% 乙醇喷洒小鼠树干, 用纸巾擦拭。把鼠标放在它的背部, 稍微倾斜到它的右侧。剪掉毛皮, 用解剖剪刀, 沿着老鼠的左边, 大约在前后两腿之间。</li>
	<li>使用镊子, 抓住腹膜, 小心做切口, 以免损伤脾脏。用镊子将脾脏取出, 放入50毫升的无菌管, 在冰上含有大约10毫升培养基。对其余的动物重复。 注: 脾脏是芸豆的颜色, 比肾脏更长、更平坦。</li>
	<li>
转移一脾脏与2毫升培养基到一个小不育培养皿。
	<ol>
<li>融汇在两个不育的磨砂片的磨砂部分之间磨碎脾脏。如果可能, 在均匀化过程中保持介质中的问题和单元格。</li>
		<li>一旦彻底均匀化, 吸管2毫升培养基中含有脾通过40µm 尼龙细胞过滤器成50毫升管。</li>
		<li>重复上述步骤, 其余的脾使用新的磨砂幻灯片, 50 毫升管与培养基和细胞过滤器。</li>
	</ol>
</li>
	<li>将8毫升的冷培养基添加到含有脾的50毫升管中的每一个, 总体积为10毫升/管。使用 hemocytometer 确定每个毫升的单元格数。</li>
	<li>增加大约 1 x 106细胞/井到6井培养板材。将单元格添加到与实验所用的不同 PAMPs 数对应的井数中, 并包括一个控制井。添加3毫升培养基或3毫升的挑战培养基到各自的水井。</li>
	<li>孵育板材在37°c 在 5% CO2为 3 h。</li>
	<li>使用连接到 P1000 微的1000µL 吸管尖, 从井底刮出细胞。用相同的1000µL 吸管尖端将含有细胞的培养基转移到15毫升管上。</li>
	<li>在室温下以 167 x g为5分钟, 通过离心法将细胞颗粒状。取出上清液, 用5毫升的 PBS 清洗细胞颗粒。</li>
	<li>第二次将细胞以 167 x g为5分钟, 取出上清, 并在细胞颗粒中加入600µL 的溶解缓冲液, 以溶解细胞。然后进行 RNA 分离。</li>
</ol>4. RNA 分离和 cDNA 合成
<ol><li>根据制造商的说明, 使用 rna 提取试剂盒将 rna 从脾中分离出来, 并执行 "选择性" 的列 DNA 消化。</li>
	<li>在 cdna 合成之前, 用 microvolume 分光光度法测定 rna 浓度, 以验证其浓度是否在 cdna 合成试剂盒的最佳 RNA 范围内 (高达2µg)。</li>
	<li>
根据制造商的指示, 使用反向转录试剂盒合成每个样品的 cDNA。在20µL 总反应量中, 每个样品使用10µL RNA。在完成反向转录 (thermocycler 运行) 增加 20 ul H2O 到每个反应.
</li>
	<li>使用 P10 或 P20 微构造标准曲线, 将一些控制样本 (例如, 5 µL 从2个样本中总共10个µL) 池中的 mRNA 放入0.5 毫升管中制备 cDNA。<ol>
<li>添加10µL 2x 逆转录酶主组合。</li>
		<li>在反向转录完成后, 将10µL 的 H2O 添加到反应管中, 这将作为标准曲线稀释系列中的起始浓度 ("1")。</li>
		<li>执行10倍稀释系列 (1 到 10-4) 通过添加45µL 水使用 P100 或 P200 微到四0.5 毫升管指定 10-1, 10-2, 10-3, 和 10-4.<ol>
<li>加入5µL 的起始浓度 "1" 进入第一管 (10-1) 使用 P20 micropipettor, 混合吹打上下几次, 然后转移5µL 从 10-1管进入管指定 10-2和混合。</li>
			<li>转移5µL 使用 P20 微从 10-2管进入管指定 10-3和混合。转移5µL 使用 P20 micropipettor 从 10-3管进入管指定 10-4和混合。</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. 定量聚合酶链反应 (qPCR)
<ol><li>通过 qPCR 测定 mRNA 水平的相对定量。在实验设置中, 选择 "定量相关标准曲线" 和 "探针" 化学。将反应量更改为10µL. 将相关信息输入到板块布局中 (如目标基因、标准曲线、记者等)。</li>
	<li>准备反应, 使其包含0.5 µL 的底漆/探针检测, 5 µL 基因表达主混合, 2 µL 的 H2O 和2.5 µL 的 cDNA (10–100 ng)。<ol>
<li>通过将前面的每种试剂 (cDNA 除外) 乘以反应的数量来准备一个主混合, 确保包括从标准曲线、负控制、重复执行每一个反应, 以及2额外的反应, 将会考虑到吹打变异。</li>
		<li>吸管7.5 µL 的准备大师混合成预先确定的水井在一个96井反应板与微或多通道吸管。吸管2.5 µL 的 cDNA 入适当井为未知数和标准, 并且2.5 µL2O 入消极控制井。</li>
		<li>包括对各种分子时钟基因的底漆/探针测定, 还包括对内源性控制 (如β肌动蛋白) 的检测。</li>
	</ol>
</li>
	<li>为了确定相对表达式值, 计算每个复制的平均相对数量, 然后对每个样本, 将目标基因的平均相对数量除以β肌动蛋白的平均相对数量。</li>
</ol>6. 统计分析
<ol><li>使用统计分析软件, 在 "列统计" 选项下, 将数据输入到程序中。选择一个单向方差分析与 Dunnett 的后专项测试, 以评估差异的平均值的时钟基因表达后玉兰挑战与控制。</li>
</ol>

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作者没有什么可透露的。

在本协议中, microvolume 分光光度计可用于量化和评估用于确定基因表达的 RNA 的纯度。核酸吸收紫外光在260毫微米, 蛋白质通常吸收光在280毫微米, 而其他潜在污染物使用在 RNA 提取过程 (例如,苯酚) 是可检测的在230毫微米。因此, 通过评估吸光度 (A) 比在260/280 毫微米 (rna 到蛋白质) 和260/230 毫微米 (rna 到非蛋白质污染物) 可以评估 rna 的质量。高质量的 RNA 在1.8–2.1 之间有 A260/280 的比值, 因为?...

主时钟在哺乳动物, 协调 24 h 振荡为几乎生理学和行为的所有方面, 位于视交叉上核之内 (SCN) 下丘脑1,2。除了调节生物过程的生物体水平, 主时钟也同步周围的蜂窝时钟在整个身体3,4,5。虽然分子时钟机械包括至少三联锁转录-平移反馈回路, 核心是由周期(Per1-3),隐花色素(Cry1-2), Bmal1,和时钟基因6,7.除了保持核心分子时钟的准确时间, 一些辅助时钟基因产品 (例如, erbα和Dbp) 也调节非时钟基因的表达,即时钟控制基因 (CCGs)6,7。
在不同的免疫组织 (如脾脏和淋巴结)8和细胞 (如B 细胞, 树突状细胞, 巨细胞)8,9描述了功能分子时钟。这些细胞通过先天免疫识别受体 (如类似收费受体 (TLRs)10) 检测和反应与病原体相关的分子模式 (PAMPs), 保守的微生物成分。到目前为止, 已经描述了13种功能性 TLRs, 它能识别细菌细胞壁成分, 鞭毛蛋白和微生物核酸10等微生物成分。玉兰, 脂多糖 (LPS), TLR4 承认革兰阴性菌的细胞壁成分, 已被证明改变昼夜节律的生物体和分子水平。例如,在体内的挑战, LPS 诱导光样相延迟的活动, 以小鼠11和导致减少时钟基因表达在 SCN 和肝脏, 由原位杂交和定量 PCR 确定,分别在大鼠12。在体内对 LPS 的挑战后, 对人外周血白细胞13和皮下脂肪组织14的分析显示, 通过 qPCR 测量的数个时钟基因的表达改变。最后,体内LPS 对人巨噬细胞和小鼠腹腔巨噬细胞的挑战, 也导致了 qPCR14测量的时钟表达的改变。
在这里, 我们描述了一个评估 PAMPs LPS, ODN1826 (含有非甲基化的合成寡核苷酸) 和热杀死李斯特菌 (HKLM) 的影响的协议, 分别由 TLR4、TLR9 和 TLR2 识别, 对小鼠脾的分子时钟基因表达。该协议包括小鼠脾切除术、脾分离和挑战、RNA 提取、cDNA 合成和 qPCR, 以评估几个时钟基因的表达。该协议允许及时获得大量的小动物或细胞操作的免疫细胞, 然后在体内使用各种 PAMPs 来挑战。分子时钟已经被证明可以调节免疫应答的各个方面8,15,16, 因此, 分子时钟的中断很可能会损害适当的时间依赖性的变化免疫应答。此外, 由于昼夜节律的中断可能导致严重的病理17,18,19,20, 它可能会感兴趣的研究人员挑战脾与广泛的分子和评估他们对时钟的影响。

<table><tbody><tr><td>Frosted slides</td><td>Fisher</td><td>12-550-343</td><td></td></tr><tr><td>Cell strainers</td><td>Fisher</td><td>22363547</td><td></td></tr><tr><td>Lipopolysaccharide&amp;nbsp;</td><td>InvivoGen</td><td>ltrl-eklps</td><td></td></tr><tr><td>ODN1826</td><td>InvivoGen</td><td>Tlrl-1826-1</td><td></td></tr><tr><td>HKLM</td><td>InvivoGen</td><td>Tlrl-hklm</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>20012-043</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104 or 74106</td><td></td></tr><tr><td>RNase-Free DNase Set</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>6-well cell culture plate</td><td>Denville</td><td>T1006</td><td></td></tr><tr><td>50 mL tubes</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>15 mL tubes</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit</td><td>ThermoFisher</td><td>4368814</td><td></td></tr><tr><td>TaqMan Gene Expression Assays b-actin</td><td>ThermoFisher</td><td>Mm00607939_s1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Per2</td><td>ThermoFisher</td><td>Mm00478113_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Rev-erba</td><td>ThermoFisher</td><td>Mm00520708_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Bmal1</td><td>ThermoFisher</td><td>Mm00500226_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Assays Dbp</td><td>ThermoFisher</td><td>Mm00497539_m1</td><td></td></tr><tr><td>qPCR machine StepOnePlus</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>ThermoFisher</td><td>4369016</td><td></td></tr><tr><td>MicroAmp Fast 96-well reaction plate (0.1 mL)</td><td>ThermoFisher</td><td>4346907</td><td></td></tr><tr><td>Statistical Analysis Software</td><td>Prism 7.0a</td><td></td><td></td></tr></tbody></table>

the use of mouse splenocytes to assess pathogen-associated molecular pattern influence on clock gene expression

从行为到基因表达, 昼夜节律调节几乎所有生理学方面。在这里, 我们提出了一个方法来挑战鼠脾与病原体相关的分子模式 (PAMPs) 脂多糖 (LPS), ODN1826 和热杀死李斯特菌, 并检查其对分子昼夜节律的影响时钟。以前, 研究重点研究了脂多糖对分子时钟的影响, 使用各种模型 (如鼠、鼠 、人) 的体内和体外方法。该协议描述了脾的分离和挑战, 以及通过定量 PCR 评估时钟基因表达后挑战的方法。这种方法不仅可以用来评估微生物成分对分子时钟的影响, 而且还能评价其他分子, 这可能改变时钟的表达。这种方法可以用来取笑玉兰受体相互作用如何影响时钟表达的分子机制。

小鼠在 ZT13 被牺牲, 脾被隔绝了和挑战前体内与 PAMPs LPS, ODN1826 或 HKLM。3小时后, RNA 被分离, qPCR 被用来评估分子时钟基因时钟、 Per2、 Dbp和 erbα的相对表达水平, 而非挑战控制细胞。在玉兰挑战之后,时钟表达式水平与控制单元中的表达式没有显著的差异 (图 1A)。Per2表达水平显著升高的细胞?...

Watch this Scientific Journal Video about The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression at JoVE.com

The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

本协议描述了一种使用小鼠脾的技术来发现改变分子时钟基因表达的病原相关分子模式。

用小鼠脾评估病原体相关分子模式对时钟基因表达的影响

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse ...

the-use-mouse-splenocytes-to-assess-pathogen-associated-molecular

Research

JoVE Journal

Genetics

7.4K 次观看.  University of Hartford. 本协议描述了一种使用小鼠脾的技术来发现改变分子时钟基因表达的病原相关分子模式。

视频: The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

科研

遗传学

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

体外生物荧光法对乳腺上皮细胞昼夜节律的表征

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

从详细的空间表达图谱的动态表达数据的时间排序

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

发育生物学

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

免疫与感染

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

鼠标型号病原体引起的慢性炎症局部及全身站点

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal allophycocyanin (APC) fluorescence intensity, are conjugated to antibodies specific to a particular analyte. Next, a selected panel of defined capture bead sets is incubated with a biological sample containing target analytes specific to the capture antibodies. A biotinylated detection antibody cocktail is added, which leads to the formation of capture bead-analyte-detection antibody sandwiches.
Finally, streptavidin-phycoerythrin (SA-PE) is added, which binds to biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the amount of bound analyte. The PE fluorescent signal of analyte-specific beads regions is quantified using flow cytometry, and the concentrations of particular analytes are determined using data analysis software and the standard curve generated in the assay.
In this experiment, we use a mouse T helper cytokine panel to simultaneously quantify the concentration of 13 separate cytokine targets in tissue culture supernatants collected from mouse splenocytes cultured under various stimulatory conditions.

This protocol describes the quantification of multiple cytokine targets simultaneously in tissue culture supernatants collected from stimulated mouse splenocytes using multiplex bead based immunoassay platform and a flow cytometer.

细胞 Bead-based 免疫分析平台对受激小鼠脾多重细胞因子的研究

Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

细菌病原体在细胞和有机水平宿主反应探针中的应用

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

体内荧光记者对小鼠视交叉上细胞核生物钟基因表达的监测

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

神经科学

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

李斯特菌单细胞基因感染后小鼠脾脏原位干扰素伽马生产的成像

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.