Name: cell type-specific gene expression profiling in the mouse liver
Uploaded: 2019-09-17T14:30:00
Description: Watch this Scientific Journal Video about Cell Type-specific Gene Expression Profiling in the Mouse Liver at JoVE.com

这项工作得到了以下赠款的支持：F31-DK113666（AWW）、K01-DK102868（AMZ）、K08-DK106478（KJW）和P30-DK050306（KJW试点赠款）。

所有涉及使用小鼠的方法都符合宾夕法尼亚大学宾夕法尼亚大学宾夕法尼亚大学动物福利办公室IACUC提供的指南。
1. 试剂制备
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赛克洛赫西米德
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<li>要使500μL为0.1克/mL环氧西米，在500μL甲醇中悬浮50毫克环己酰胺。 警告：环己酰胺对环境毒性极强，可引起先天性畸形。所有含有环己酰胺的废物和缓冲液应收集，以便妥善处置。 注...

<ol>
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J., 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=A+facile+method+for+somatic,+lifelong+manipulation+of+multiple+genes+in+the+mouse+liver.">A facile method for somatic, lifelong manipulation of multiple genes in the mouse liver.</a> Hepatology. 47 (5), 1714-1724 (2008).</li><li>Wang, A. W., 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=TRAP-seq+identifies+cystine/glutamate+antiporter+as+a+driver+of+recovery+from+liver+injury.">TRAP-seq identifies cystine/glutamate antiporter as a driver of recovery from liver injury.</a> The Journal of Clinical Investigation. 128 (6), 2297-2309 (2018).</li><li>Heiman, M., Kulicke, R., Fenster, R. J., Greengard, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+mRNA+purification+by+translating+ribosome+affinity+purification+(TRAP).">Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP).</a> Nature Protocols. 9 (6), 1282-1291 (2014).</li><li>Agilent Technologies. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Agilent RNA 6000 Pico: User Manual. , (2016).</li><li>NEBNext. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NEBNext Ultra RNA Library Prep Kit for Illumina: User Manual. , (2018).</li><li>NanoDrop. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2000/2000c Spectrophotomerter: User Manual. , (2009).</li><li>Liu, J., 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=Cell-specific+translational+profiling+in+acute+kidney+injury.">Cell-specific translational profiling in acute kidney injury.</a> The Journal of Clinical Investigation. 124 (3), 1242-1254 (2014).</li><li>Lu, S. 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TRAP-seq是一种通过表位标记核糖体翻译mRNA的细胞类型特异性分离技术，它提供了FACS方法的替代方法，因为它规避了FACS9的时间要求等限制。相反，TRAP允许从散装组织中直接快速有效地分离RNA，帮助避免基因表达的任何改变。TRAP-seq特别适合用于重新填充Fah-/-小鼠肝脏，因为去除尼西酮后的肝细胞扩张是细胞自主的，并且通过集成功能使肝细胞子集的基因表达分析?...

肝脏作为脊椎动物的主要代谢器官，负责葡萄糖平衡、血清蛋白合成、胆汁酸分泌、异种生物代谢和排毒。肝脏具有非凡的能力，在接触毒素时再生受伤的帕伦奇马，以防止立即肝衰竭1。然而，再生失败可能发生在对乙酰氨基酚或酒精过量消费的设置，这可能导致急性肝衰竭2。此外，病毒性肝炎感染、脂肪肝、脂肪性肝炎引起的慢性肝损伤可引起肝纤维化、肝硬化、肝细胞癌3。治疗终末期肝病的唯一治疗方法是移植，但受器官短缺的限制，无法对所有患者进行有效治疗。因此，更好地了解毒性肝损伤后的恢复过程对于开发治疗以刺激足以挽救患病器官功能的再生至关重要。
肝再生研究应用最广的模型系统是啮齿动物的局部肝切除术，其中大部分肝脏被切除以刺激肝细胞快速扩张5。然而，部分肝切除术不重述肝细胞扩张后，毒性肝损伤，由于缺乏免疫细胞渗透和肝细胞坏死经常观察到在急性肝损伤设置在人类6。一个更合适的系统来模拟这种形式的器官更新是Fah-/-小鼠，它缺乏功能性紫甲酸氢酶（FAH）所需的适当的酪氨酸代谢，并开发严重的肝脏损伤，导致死亡7。通过饮用水中的药物尼他酮治疗，这些小鼠可以无限期地保持健康状态。或者，FAH表达可以通过转基因递送到肝细胞的子集来恢复，在消除尼西酮8时，肝细胞将膨胀以重新填充肝脏。
为了分析重新填充肝细胞的基因表达变化，需要一种工具，专门分离Fah-/-小鼠中复制的肝细胞，而不受邻近受伤的肝细胞和其他细胞类型的污染。不幸的是，肝细胞的荧光辅助细胞分拣（FACS）很困难，因为（1）肝细胞再生的脆弱性导致肝灌注后恢复不良，（2）复制肝细胞的大小变化很大，使得隔离纯种群由FACS困难，和（3）从肝脏灌注到RNA分离的手术时间大于2小时，因此基因表达谱可能在采集样品之前发生重大人工变化。
或者，表位标记核糖体的表达，专门在重新填充肝细胞允许快速分离积极翻译mRNA结合核糖体结合在器官收获后立即亲和纯化，与散装肝脏组织莱沙。在这里，我们描述了一个协议，以执行翻译核糖体亲和纯化（TRAP）10，然后是高通量RNA测序（TRAP-seq），在Fah-/- 中特别分离和剖分mRNA，以重新填充肝细胞- /-鼠标9.绿色荧光蛋白标记核糖体蛋白（GFP：RPL10A）与FAH的共聚体蛋白（GFP：RPL10A）与FAH的共聚体结合，允许通过含有GFP：RPL10A的聚体结合的翻译mRNA进行亲和纯化。这种方法避免了任何细胞分离步骤，如肝脏灌注，以分离脆弱的再生肝细胞。相反，它利用整个器官组织和抗体的解毒，快速从靶细胞中快速提取RNA。最后，通过TRAP-seq分离大量、高质量的mRNA，使测序分析等下游应用能够分析基因表达在再填充过程中的动态变化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL Tissue Grinder, Potter-Elv, Coated</td>
			<td>DWK Life Sciences (Wheaton)</td>
			<td>358007</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolutely RNA Miniprep Kit</td>
			<td>Agilent</td>
			<td>400800</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP antibodies</td>
			<td>Memorial Sloan-Kettering Antibody &#38; Bioresource Core</td>
			<td colspan="2">GFP Ab #19C8 and GFP Ab #19F7</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, IgG-Free, Protease-Free</td>
			<td>Jackson ImmunoResearch</td>
			<td>001-000-162</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Millipore Sigma</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxycholic acid, DOC</td>
			<td>Millipore Sigma</td>
			<td>D2510</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose, Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D16</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol</td>
			<td>Millipore Sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads MyOne Streptavidin T1</td>
			<td>Thermo Fisher Scientific</td>
			<td>65602</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Petri Dishes with Clear Lid</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (10x), calcium, magnesium, no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>14065-056</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, 1M Solution, pH 7.3, Molecular Biology Grade, Ultrapure, Thermo Scientific</td>
			<td>Thermo Fisher Scientific</td>
			<td>AAJ16924AE</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride, MgCl2&#160;</td>
			<td>Millipore Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A452</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000/2000c Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>VV-83061-00</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBNext Poly(A) mRNA Magnetic Isolation Module</td>
			<td>New England BioLabs</td>
			<td>E7490S</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBNext Ultra RNA Library Prep Kit for Illumina</td>
			<td>New England BioLabs</td>
			<td>E7530S</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonylphenyl polyethylene glycol, NP-40. IGEPAL CA-630</td>
			<td>Millipore Sigma</td>
			<td>I8896</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free Water, not DEPC-Treated</td>
			<td>Ambion</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>Overhead Stirrer</td>
			<td>DWK Life Sciences (Wheaton)</td>
			<td>903475</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Buffer (10x), pH 7.4</td>
			<td>Ambion</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Recombinant Protein L, Biotinylated</td>
			<td>Thermo Fisher Scientific</td>
			<td>29997</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride, KCl</td>
			<td>Millipore Sigma</td>
			<td>P4504</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA 6000 Pico Kit &#38; Reagents</td>
			<td>Agilent</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaseZap RNase Decontamination Solution</td>
			<td>Invitrogen</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>RNasin Ribonuclease Inhibitors</td>
			<td>Promega</td>
			<td>N2515</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide, NaN3</td>
			<td>Millipore Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate, NaHCO3</td>
			<td>Millipore Sigma</td>
			<td>S6297</td>
			<td></td>
		</tr>
		<tr>
			<td>SUPERase&#183;In RNase Inhibitor</td>
			<td>Invitrogen</td>
			<td>AM2694</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell type-specific gene expression profiling in the mouse liver

受伤后肝脏重新填充是哺乳动物的一个关键特征，它可以防止在接触环境毒素后立即导致器官衰竭和死亡。更深入地了解重放期间发生的基因表达变化，有助于确定治疗目标，促进损伤环境中肝功能的恢复。然而，由于缺乏细胞标记、细胞数量有限以及这些细胞的脆弱性，特别分离重新造血肝细胞的方法受到抑制。结合Fah-/-小鼠模型，在肝损伤设置中重述再填充，使核糖体亲和纯化（TRAP）技术得以转化，从而允许重新填充的基因表达分析肝 细胞。使用TRAP，细胞类型特异性翻译mRNA被快速有效地分离。我们开发了一种方法，利用TRAP与基于亲和力的分离，从肝细胞翻译mRNA，选择性地表达绿色荧光蛋白（GFP）标记核糖体蛋白（RP），GFP：RPL10A。TRAP 绕用荧光激活细胞分类所需的长时间，可改变基因表达轮廓。此外，由于只有重新填充的肝细胞表达GFP：RPL10A融合蛋白，分离的mRNA没有来自周围受伤的肝细胞和肝脏中其他细胞类型的污染。亲和力纯化的mRNA是高质量的，允许下游PCR或高通量测序基于基因表达的分析。

为了在重新填充Fah-/-小鼠的肝细胞中分析基因表达，Gfp：Rpl10a融合和法赫转基因在含有质粒的转位子8（TRAP载体）中共同传递到肝脏，流体动力学注射 （图 1A）去除尼西酮诱导毒性肝损伤，为肝细胞产生选择压力，使其能稳稳地表达FAH，以重新填充受伤的帕伦奇马9。免疫?...

Watch this Scientific Journal Video about Cell Type-specific Gene Expression Profiling in the Mouse Liver at JoVE.com

Cell Type-specific Gene Expression Profiling in the Mouse Liver

翻译核糖体亲和性纯化（TRAP）可实现细胞类型特异性翻译mRNA的快速和高效分离。在这里，我们演示了一种将水动力尾静脉注射与肝脏再填充小鼠模型和TRAP相结合的方法，以检查重新填充肝细胞的表达特征。

小鼠肝脏中细胞类型特异性基因表达分析

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

使用 TRAP-seq，我们可以从特定的细胞类型中分离出RNA，例如在受伤肝脏中重新填充的肝细胞，以分析这些细胞中发生的基因表达变化。它不需要任何步骤来去除组织中不需要的细胞。细胞特异性RNA通过从整个器官的亲和纯化分离。这项技术帮助我们确定特定细胞对损伤的反应，这有助于确定对抗肝病的新策略或药物。这可以用来识别肝细胞对不同类型的肝损伤的反应。目前，有很少的选择，严重的急性肝损伤，这种方法将有助于线索，我们到新的治疗方法。这种方法起源于大脑。在肝脏中，我们设想它可以用来检查各种细胞类型的动态变化，肝细胞，胆道细胞，库普弗细胞和内皮细胞，举几例。演示程序将是琥珀王，一个研究生和我的门名。首先，通过温和的移液重新悬浮磁珠。将磁支架上的珠子收集超过一分钟，然后取出上经剂。然后，从磁性支架上取出微离心管，加入一毫升PBS，然后上下移液清洗珠子。将管子放回磁性支架上超过一分钟，收集磁珠并取出 PBS。要准备蛋白质L涂层珠子，在重新悬浮和洗涤的磁珠中加入16微升的生物素化蛋白L，如果使用1.5毫升微离心管，则加入1X PBS，使最终体积为1毫升，如果使用2毫升微离心管，或1.5毫升。在室温下，用生物素化蛋白L孵育磁珠35分钟。然后，将管子放在磁性支架上超过一分钟，取出上清液以收集蛋白质 L 涂层珠。从磁性支架上取出微离心管，用 3%BSA 缓冲液加入一毫升 PBS，然后轻轻移液至少五次，以清洗蛋白 L 涂层珠子。再次，将管子放在磁性支架上超过一分钟，取出上经剂以收集涂层珠子。再重复 BSA 洗涤四次。接下来，将50微克的GFP抗体19C8和GFP抗体19F7加入蛋白质L涂层珠，在4摄氏度的管旋转器上孵育1小时。要收集亲和力矩阵，请将管子放在磁性支架上超过一分钟，然后取出上经剂。将管子从磁性支架上拿开，加入一毫升低盐缓冲液。轻轻上下移液，清洗亲和力矩阵，再重复低盐缓冲液再洗两次。将珠子重新填充在200微升的低盐缓冲液中，使200微升的亲和力矩阵。首先，设置组织研磨机，使PTFE玻璃管在均质过程中可以放在冰上。将四毫升冷解液缓冲液填充到PTFE玻璃管中。将肝脏放在培养皿上。使用刀分离200至500毫克的肝脏碎片，并进入PTFE玻璃管。将剩余的肝脏组织放入预冷的微离心管中，并在液氮中闪冻。在电机驱动的均质器中均匀化样品，从 300 rpm 开始至少五次冲程，将肝细胞与肝脏结构分离。每次降低玻璃管。防止呼吸，这可能会导致蛋白质变性，保持溶液下面的害虫。然后，将转速提升至 900 rpm，使肝脏组织完全均匀，至少进行 12 次全冲程。将落酸转移到标签和预冷却管中，每个管不超过一毫升的落酸。为了开始核解解，将肝脏在4摄氏度下以2000次g离心，10分钟，然后将上流液转移到冰上一个新的预冷却微离心管。将10%NP-40的上半液体积的1/9加入冰上微离心管中，轻轻反转管，混合溶液。使用微型离心机快速旋转微离心管，加入 10% DOC 的 1/9 样品体积。通过轻轻反转微离心管混合，快速旋转微离心管并在冰上孵育五分钟。将2万次甘酸的核酸盐在4摄氏度下离心10分钟，然后将上流水液转移到新的冰上冷却前的微离心管上。对于每根管子，将上每根液的1%作为免疫前沉淀控制。将免疫前沉淀控制装置在四摄氏度的管旋转器上放置一夜。格外小心，通过移液轻轻重新暂停亲和矩阵，然后向每个样品添加 200 微升。在四摄氏度下孵育赖酸盐过夜，在管旋转器上轻轻混合。要分离RNA，请快速旋转微型离心机中含有裂酸的管，然后通过放置在磁架上至少放置一分钟来收集珠子。在额外的微离心管中收集上流液。在每个管中加入一毫升新鲜高盐缓冲液，然后轻轻移液至少五次，而不产生气泡。将磁性支架上的珠子收集超过一分钟，然后取出上一液。再重复四次洗涤步骤。然后，从磁性支架上取出微离心管，在室温下放置五分钟以预热。将珠子用0.7微升β-墨二甲醇在100微升的解解缓冲液中，从亲和力基质中释放结合RNA。以最高速度旋转管至少五秒钟，然后快速向下旋转。然后，在室温下孵育管子10分钟。将管子放在磁性支架上至少一分钟，然后收集上经剂，然后根据试剂盒中的RNA纯化方案立即进行RNA清理。为了达到分离RNA的最大质量，执行所有可选步骤，包括DNase消化和所有RNA洗脱步骤，包括建议将洗脱缓冲液预热至60摄氏度。在这项研究中，GFP-L10A融合和法赫转基因通过流体动力学注射在含有转波子质粒的诱捕载体中共同传递到肝脏。尼西诺酮的去除引起肝毒性损伤。免疫荧光染色证实了FAH和GFP-L10A融合蛋白的共表达，以及肝脏再填充两周后重新填充的肝细胞。由于所有肝细胞在AAV8-TBG-Cre注射后都表达GFP-L10A，因此该基质样品产生最高产量的融合蛋白。相反，在野生型对控中，几乎没有任何RNA被检测到，这些对控制没有GP-L10A转基因，这表明 TRAP程序非常具体，背景较低。当 TRAP 用于肝组织使用 GFP-L10A 转导肝细胞重新填充时，获得了丰富的高质量RNA。相比之下，没有通过生物分析器检测出对阴性对照样品的RNA微量。与静态肝细胞相比，Gsta1表达在重新填充肝细胞时诱导了十倍以上，而由于缺乏输入RNA，未检测到野生型小鼠的 TRAP 隔离 RNA 的阈值周期值。当组织均质时，确保缓慢移动害虫，以防止气化。隔离RNA后，可以进行高通量测序或qPCR分析基因表达。使用 TRAP-seq，我们现在有一种方法，专门分离RNA从重新填充的肝细胞，并分析在肝脏重新填充期间基因表达的变化。这使我们能够识别在这个过程中改变的基因和治疗肝损伤的潜在治疗目标。环氧胺和DTT，存在于几个缓冲液中，都是有毒的。应根据体制准则收集和丢弃废物。

cell-type-specific-gene-expression-profiling-in-the-mouse-liver

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Biology

Unit 1

Gene Expression

SCIENTISTS IN ACTION

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

科研

生物学

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髓电

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

激光显微切割的从细胞的一个子集基因表达分析中的应用果蝇永光盘

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Heterokaryon Technique for Analysis of Cell Type-specific Localization

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling. These proteins display a diverse array of cellular functions including nuclear import/export of RNA and protein, transcriptional regulation, and apoptosis. Interestingly, major cellular reorganizations including cell division, differentiation and transformation, often involve such activities3,4,8,10. The detailed study of these proteins and their respective regulatory mechanisms can be challenging as the stimulation for these localization changes can be elusive, and the movements themselves can be quite dynamic and difficult to track. Studies involving cellular oncogenesis, for example, continue to benefit from understanding pathways and protein activities that differ between normal primary cells and transformed cells6,7,11,12. As many proteins show altered localization during transformation or as a result of transformation, methods to efficiently characterize these proteins and the pathways in which they participate stand to improve the understanding of oncogenesis and open new areas for drug targeting.
Here we present a method for the analysis of protein trafficking and shuttling activity between primary and transformed mammalian cells. This method combines the generation of heterokaryon fusions with fluorescence microscopy to provide a flexible protocol that can be used to detect steady-state or dynamic protein localizations. As shown in Figure 1, two separate cell types are transiently transfected with plasmid constructs bearing a fluoroprotein gene attached to the gene of interest. After expression, the cells are fused using polyethylene glycol, and protein localizations may then be imaged using a variety of methods. The protocol presented here is a fundamental approach to which specialized techniques may be added.

A flexible and efficient method for the characterization of cell type-specific protein localization and nucleocytoplasmic shuttling is described. This heterokaryon approach uses fluorescently-labeled fusion proteins to image protein localizations after cell fusion. The protocol is amenable to steady-state localizations or more dynamic determinations based on live cell imaging.

异核技术的细胞类型特异性定位分析

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

高分辨率基因表达谱RNA的合成，加工和衰变的新转录RNA在细胞培养的代谢标记

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...