Name: the use of mouse splenocytes to assess pathogen-associated molecular pattern influence on clock gene expression
Uploaded: 2018-07-24T15:00:00
Description: 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

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 (...

<ol>
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Res. 145, 5-12 (2008).</li><li>Haimovich, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+endotoxin+synchronizes+and+suppresses+clock+gene+expression+in+human+peripheral+blood+leukocytes.">In vivo endotoxin synchronizes and suppresses clock gene expression in human peripheral blood leukocytes.</a> Crit. Care Med. 38, 751-758 (2010).</li><li>Curtis, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+control+of+innate+immunity+in+macrophages+by+miR-155+targeting+Bmal1.">Circadian control of innate immunity in macrophages by miR-155 targeting Bmal1.</a> Proc. Natl. Acad. Sci. U. S. A. 112, 7231-7236 (2015).</li><li>Silver, A. C., Arjona, A., Walker, W. 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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 border="0" cellpadding="0" cellspacing="0" style="width:796px;" width="795">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:343px;">Frosted slides</td>
			<td style="width:116px;">Fisher</td>
			<td style="width:137px;">12-550-343</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell strainers</td>
			<td>Fisher</td>
			<td align="right">22363547</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipopolysaccharide&#160;</td>
			<td>InvivoGen</td>
			<td>ltrl-eklps</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ODN1826</td>
			<td>InvivoGen</td>
			<td>Tlrl-1826-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HKLM</td>
			<td>InvivoGen</td>
			<td>Tlrl-hklm</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Gibco</td>
			<td>20012-043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104 or 74106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase-Free DNase Set</td>
			<td>Qiagen</td>
			<td align="right">79254</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well cell culture plate</td>
			<td>Denville</td>
			<td>T1006</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 ml tubes</td>
			<td>Corning</td>
			<td align="right">352070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 ml tubes</td>
			<td>Corning</td>
			<td align="right">352097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Capacity cDNA Reverse Transcription Kit</td>
			<td>ThermoFisher</td>
			<td align="right">4368814</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays b-actin</td>
			<td>ThermoFisher</td>
			<td>Mm00607939_s1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Per2</td>
			<td>ThermoFisher</td>
			<td>Mm00478113_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Rev-erba</td>
			<td>ThermoFisher</td>
			<td>Mm00520708_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Bmal1</td>
			<td>ThermoFisher</td>
			<td>Mm00500226_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Dbp</td>
			<td>ThermoFisher</td>
			<td>Mm00497539_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">qPCR machine StepOnePlus</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Master Mix</td>
			<td>ThermoFisher</td>
			<td align="right">4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroAmp Fast 96-well reaction plate (0.1 ml)</td>
			<td>ThermoFisher</td>
			<td align="right">4346907</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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

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Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Production and Purification of Baculovirus for Gene Therapy Application

杆状病毒基因治疗应用的生产与纯化

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

血栓烷 A2 受体基因 C924T 多态性的研究方法

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...