通过赠款，这项工作是由威康信托基金资助的APM [076566/Z/05/Z]，JCK [074318]和威康信托中心人类遗传学的核心奖[075491/Z/04]。

1）细胞培养<ol><li>选择感兴趣的基因表达的细胞类型。 </li><li>选择DNA风险的变种和转录编码内感兴趣的基因多态性（图1）杂合子的细胞株。 </li><li>文化所选单元格的行根据供应商的规格，并准备分别为2 × 10厘米的培养皿中，DNA和RNA的提取。 </li><li>当细胞达到80％汇合时启动程序，收获细胞，DNA和RNA的隔离遵循下述步骤或使用市售的试剂盒。 </li></ol> 2）DNA的提取<ol><li>从盘取出介质，用PBS清洗，并添加到菜500μL裂解液（0.6％SDS，氯化钠100毫米，50毫米的Tris，20 mM的EDTA和50μg/ mL的RNA酶A）。 </li><li>岩石板，轻轻地在室温下20分钟。 </li><li>在37 ° C过夜，刮的离心管中，并培育成的细胞裂解液。 </li><li>加入蛋白酶K至终浓度为100微克/毫升和37℃过夜孵育。 </li><li>与等体积酚 - 氯仿的pH值8，重复提取DNA，然后与等体积的氯仿/异戊醇提取。 </li><li> 1 / 10体积3 M氯化钠和2.5卷100％的乙醇沉淀的DNA。 </li><li>留在冰浴5分钟，然后在30分钟13000转，旋转4 ° C。 </li><li>用70％乙醇清洗DNA，使空气干燥，在200μL的TE悬浮。 </li><li>在65℃孵育10分钟，然后在室温下2天离开。 </li><li>保存在4 ° C或长期在-20 ° C </li></ol> 3）RNA的提取<ol><li>从盘取出介质，用PBS清洗，添加1毫升的Trizol，并培育在室温下10分钟。 </li><li>刮下的细胞，吸离心管几次，转让和孵育5分钟。 </li><li>以10,000 rpm 10分钟旋转4 ° C。 </li><li>转移在一个新的管的上清，加入200μl氯仿。 </li><li>猛烈摇动并以10,000 rpm 10分钟旋转4 ° C。 </li><li>在一个新的管水阶段转移和70％的乙醇添加量相等。 </li><li>负载的解决方案，以1或2（上卷而定）的RNeasy列和旋转的最大速度为30秒。 </li><li>从这里遵循的RNeasy试剂​​盒（Qiagen）的指令。 </li></ol> 4）核酸样品制备<ol><li>量化的DNA和RNA的浓度，使用NanoDrop分光光度计（Thermo Scientific的）。 </li><li>准备从500-1000吴RNA根据制造商的指示使用随机decamers和标三反转录酶（Invitrogen公司）的cDNA。 </li></ol> 5）PCR引物延伸实验<ol><li>设计两对PCR引物，每个DNA分子标记，基因组DNA扩增的一对，另一对cDNA扩增（图2）。广场正向和反向在不同的外显子cDNA扩增引物，以避免基因污染。设计引物，在100-500 bp的长度范围内获得的PCR产物。 </li><li>准备10μL包括0.5 U Immolase TAQ（Bioline）0.8毫米dNTPs，MgCl 2的 2毫米，0.2米的引物和20纳克cDNA或DNA的PCR反应。每个样品的扩增4个独立的反应。 </li><li>每个引物对保持线性相位的扩增循环数的优化条件下运行的PCR反应。 </li><li>取出孵化与核酸外切酶的PCR产物，PCR引物和dNTPs过剩我和虾碱性磷酸酶（SAP）的，在37 ° 20分钟。 </li><li>现货在384孔板每两次PCR产物，因此，8测量每个样品。 </li><li>设计与实验设计（Sequenom公司）软件的延伸引物，使相同的引物可用于基因组和cDNA PCR产物。引物长度在14-18 bp和延伸组合，包括3ddNTPs组合（图2）的范围内指定。 </li><li>开展引物延伸反应和MALDI-TOF/MS分析，根据制造商的指示使用MassARRAY系统（Sequenom公司）。 </li><li>一个典型的引物延伸反应，包括2分钟94步° C，5秒94 ° C，40个周期为5秒，5秒，52℃72℃ </li><li>引物延伸产品SpectroCLEAN树脂清洗，并转移到芯片（芯片）SpectroPOINT纳升级饮水机。 </li><li>扩展的寡核苷酸使用SpectroREADER质谱仪的MALDI - TOF量化。 </li></ol> 6）统计分析<ol><li>检查MALDI-TOF/MS与实验的总质量MassARRAY导电型测量仪软件结果。 </li><li>提取Allelotyping的报告，其中包括峰面积数据。 </li><li>对于每个单独的频谱计算等位基因1和等位基因2（峰面积之比）的峰面积之间的比率。 </li><li>对于每个cDNA样本推导出一个峰面积比值的均值和标准差，使用相应的功能，在Excel中，横跨8测量。 </li><li>推导出整个基因组DNA的8 measurments相同的平均峰面积之比。 </li><li>由基因组的平均比率评估偏离预期的1:1的比例分成的cDNA的平均比例。 </li></ol> 7）实验控制<ol><li>为了排除实验文物，至少有三次重复实验。 </li><li>只要有可能，额外的DNA分子标记的设计分析。 </li><li>设计多重PCR引物对和延伸引物退火正向和反向链。 </li><li>每当可能的测试，作为阴性对照细胞株，为DNA分子标记杂合，但不进行风险与表型相关的DNA变异（图1和图3。） </li></ol> 8）代表性的成果等位基因特异性表达分析的一个例子是在质谱痕迹的例子图3所示。该图显示了从4等位基因特异性引物延伸产品4种不同的模板进行分析的结果。顶部的图形表示，从两个不同的细胞系，这都是一个转录的编码基因多态性杂合子基因组DNA的分析得到的结果。然而，只有细胞系，一个是风险的DNA变异（图1）杂合子。较低的图形代表从相同的细胞系产生的cDNA进行分析得出的结果。作为一个实验神器的第一个高峰期总是高于的第二个高峰期，即使在基因组分析。因此，从基因组分析的结果用于正常化的cDNA数据。正常化后，等位基因的比例从1细胞株显着不同（其中的第二个高峰期出现低比其他光谱），虽然这是非常密切的细胞株为1 B.数据表明，细胞系一个带有DNA的变种，测量的第二个高峰期，从而降低了在分析基因表达的等位基因的阶段。细胞B线提供了十分便利的阴性对照。 <img src="/files/ftp_upload/2279/2279fig1.jpg" alt="图1" /> 图1。 A和B细胞株细胞系的选择策略 。编码一个转录标记（三角形）的杂合子，但只有一个是对风险的DNA变异（圆圈）杂合子的细胞系。蓝色的风险变异等位基因关联（直接的效果，或在与实际的功能变异密切相关，因为）的转录水平较低。 <img src="/files/ftp_upload/2279/2279fig2.jpg" alt="图2" /> 图2。等位基因特异性引物延伸实验。这个数字表明双方的cDNA和基因组DNA（左）（右）模板进行的实验过程的工作流程。 PCR引物（灰色矩形）放大一个杂合的编码基因多态性（三角形）。为了避免基因污染PCR引物退火序列放置在不同的外显子（淡蓝色酒吧）cDNA为模板进行扩增时。 PCR产物为模板，然后用一个引物延伸反应进行扩展底漆，退火一个基地旁边的多态性，三个双脱氧适当搭配（ddNTPs）终结符（正方形）和一个脱氧核苷酸（圈），以延长一个或两个的基础上底漆。所产生的引物延伸产品，允许不同的分离和MALDI - TOF质谱定量群众。相应的蓝色基因的DNA标记的产品的数量是相对较低的红色等位基因。 <img src="/files/ftp_upload/2279/2279fig3.jpg" alt="图3" /> 图3。一个等位基因特异性表达检测结果。数字显示质谱分析进行基因组DNA和细胞株的基因A和细胞B线（图1） 。

<ol>
	<li>Cookson, W., Liang, L., Abecasis, G., Moffatt, M., Lathrop, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+complex+disease+traits+with+global+gene+expression.">Mapping complex disease traits with global gene expression.</a> Nat Rev Genet. 10, 184-194 (2009).</li><li>Singer-Sam, J., LeBon, J. M., Dai, A., Riggs, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive,+quantitative+assay+for+measurement+of+allele-specific+transcripts+differing+by+a+single+nucleotide.">A sensitive, quantitative assay for measurement of allele-specific transcripts differing by a single nucleotide.</a> PCR Methods Appl. 1, 160-163 (1992).</li><li>Yan, H., Yuan, W., Velculescu, V. E., Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+variation+in+human+gene+expression.">Allelic variation in human gene expression.</a> Science. 297, 1143-11 (2002).</li><li>Ding, C., Cantor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+gene+expression+analysis+technique+using+competitive+PCR+and+matrix-assisted+laser+desorption+ionization+time-of-flight+MS.">A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS.</a> Proc Natl Acad Sci U S A. 100, 3059-3064 (2003).</li><li>Gabriel, S., Ziaugra, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNP+genotyping+using+Sequenom+MassARRAY+7K+platform.">SNP genotyping using Sequenom MassARRAY 7K platform.</a> Curr Protoc Hum Genet Chapter. Chapter 2 (Unit 2), 12-12 (2004).</li><li>Lo, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+variation+in+gene+expression+is+common+in+the+human+genome.">Allelic variation in gene expression is common in the human genome.</a> Genome Res. 13 (8), 1855-1862 (2003).</li><li>Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression-based+discovery+of+variation+in+the+human+glutathione+S-transferase+M3+promoter+and+functional+analysis+in+a+glioma+cell+line+using+allele-specific+chromatin+immunoprecipitation.">Expression-based discovery of variation in the human glutathione S-transferase M3 promoter and functional analysis in a glioma cell line using allele-specific chromatin immunoprecipitation.</a> Cancer Res. 65, 99-104 (2005).</li><li>Knight, J. C., Keating, B. J., Rockett, K. A., Kwiatkowski, D. P. <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+characterization+of+regulatory+polymorphisms+by+allele-specific+quantification+of+RNA+polymerase+loading.">In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading.</a> Nat Genet. 33 (4), 469-475 (2003).</li><li>Pastinen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+genetic+and+epigenetic+variation+affecting+human+gene+expression.">A survey of genetic and epigenetic variation affecting human gene expression.</a> Physiol Genomics. 16 (2), 184-193 (2004).</li><li>Francks, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+77-kilobase+region+of+chromosome+6p22.2+is+associated+with+dyslexia+in+families+from+the+United+Kingdom+and+from+the+United+States.">A 77-kilobase region of chromosome 6p22.2 is associated with dyslexia in families from the United Kingdom and from the United States.</a> Am J Hum Genet. 75 (6), 1046-1058 (2004).</li><li>Paracchini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chromosome+6p22+haplotype+associated+with+dyslexia+reduces+the+expression+of+KIAA0319,+a+novel+gene+involved+in+neuronal+migration.">The chromosome 6p22 haplotype associated with dyslexia reduces the expression of KIAA0319, a novel gene involved in neuronal migration.</a> Hum Mol Genet. 15 (10), 1659-1666 (2006).</li></ol>

这种方法使体内等位基因特异性转录水平的差异，评估评价疾病相关基因表达的DNA变异的影响。在这个协议中的相对转录丰度的MALDI - TOF，但其他技术允许，如TaqMan探针6,7，等位基因特异的定量测量，都可以使用。这种方法的主要限制是转录编码标记的可用性。这里描述的方法原则的基础上，但利用不同类别的多态性，已描述。 haploChip检测措施，使用1 KB的转录的开始或结束一个基因，这将是在与特定RNA 聚合酶 II 8的抗体染色质免疫沉淀隔离材料的网站内的标记的等位基因特异性表达。此外，内含子单核苷酸多态性模板时，可以使用异RNA 的 9 。 这里描述具体的协议，与haploChip检测相结合，已成功地用于确定为读写障碍的候选基因（或阅读障碍）。最初遗传关联研究，发现与诵读困难和这是跨越3个基因的10相关的DNA序列。等位基因特异的基因表达检测结果显示，在存在的诵读困难相关的DNA变异的表达变化，发现只有三个基因中的一个，但不是其他两个11。

<table border="1">
	<thead>
		<tr>
			<th>Material Name</th>
			<th>Type</th>
			<th>Company</th>
			<th>Catalogue Number</th>
			<th>Comment</th>
		</tr>
	</thead>
	<tbody>
		<tr>
			<td>PBS</td>
			<td>&nbsp;</td>
			<td>Sigma</td>
			<td>P-4417-100TAB</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>&nbsp;</td>
			<td>Biorad</td>
			<td>161-0416</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>&nbsp;</td>
			<td>VWR</td>
			<td>27788.366</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>&nbsp;</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>&nbsp;</td>
			<td>Sigma</td>
			<td>E5134-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>&nbsp;</td>
			<td>Qiagen</td>
			<td>19101</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>&nbsp;</td>
			<td>Sigma</td>
			<td>P2308-500MG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phenol: chloroform</td>
			<td>&nbsp;</td>
			<td>Sigma</td>
			<td>77617</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>&nbsp;</td>
			<td>VWR</td>
			<td>100777C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>&nbsp;</td>
			<td>Sigma</td>
			<td>32221-2.5L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>&nbsp;</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNeasy kit</td>
			<td>&nbsp;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SuperScript III Reverse Transcriptase</td>
			<td>&nbsp;</td>
			<td>Invitrogen</td>
			<td>18080-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Immolase Taq</td>
			<td>&nbsp;</td>
			<td>Bioline</td>
			<td>BIO-21048</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>dNTP</td>
			<td>&nbsp;</td>
			<td>Sigma</td>
			<td>DNTP100A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Exonuclease 1</td>
			<td>&nbsp;</td>
			<td>NEB</td>
			<td>M0293S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Shrimp Alkaline Phosphatase</td>
			<td>&nbsp;</td>
			<td>GE Healthcare</td>
			<td>E70092Z</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SpectroCLEAN resin</td>
			<td>&nbsp;</td>
			<td>Sequenom</td>
			<td>10053</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MassEXTEND ddNTP/dNTP mix </td>
			<td>&nbsp;</td>
			<td>Sequenom</td>
			<td>&nbsp;</td>
			<td>Varies according to assay design</td>
		</tr>
		<tr>
			<td>MassEXTEND thermosequenase </td>
			<td>&nbsp;</td>
			<td>Sequenom</td>
			<td>10052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chilled microcentrifuge</td>
			<td>&nbsp;</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NanoDrop Spectrophotometer</td>
			<td>&nbsp;</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SpectroPOINT nanolitre dispenser</td>
			<td>&nbsp;</td>
			<td>Sequenom</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SpectroREADER mass spectrometer</td>
			<td>&nbsp;</td>
			<td>Sequenom</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

an allele-specific gene expression assay to test the functional basis of genetic associations

与常见的复杂特征显着的遗传协会的数量不断增加。然而，这些协会的大多数并没有被理解在分子水平上。介导的DNA变异的表型的影响的机制之一是，这已被证明特别是对复杂性状1相关的基因表达。 这种方法测试蜂窝上下文特定的DNA序列，基因表达的影响。其原理是相对丰度测量所产生的一个基因的两个等位基因，分析细胞进行与疾病（风险变种）2,3相关的DNA序列的一个副本成绩单。因此，这种方法使用的细胞应该满足了两个基本基因型要求:他们必须为DNA风险的变种和DNA分子标记，通常的编码基因多态性，基于他们的染色体的起源（图1）可以区分成绩单的杂合子。 DNA风险的变种和DNA标记不需要具有相同的等位基因频率，但阶段（haplotypic）的遗传标记的关系，需要理解。同样重要的是，选择感兴趣的基因的细胞类型，表达。此协议专指通过从成纤维细胞中提取核酸的过程，但该方法是同样适用于其他细胞类型，包括原代细胞。 从选定的细胞株DNA和RNA的提取和cDNA的生成。 DNA和cDNA进行了分析与设计目标编码DNA标记4，一个引物延伸实验。引物延伸实验进行使用MassARRAY（Sequenom公司）5平台，根据制造商的规格。引物延伸产品，然后分析了基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF/MS）时间。由于选定的标记是杂合子，他们将生成两个山峰上的MS情景。每个峰的面积是成正比的转录丰度和可与MassARRAY导电型测量仪软件功能生成一个等位基因的比例（等位基因1:等位基因2）测量计算。等位基因的cDNA获得的比例是使用从基因组DNA，其中等位基因的比例预计为1:1，以正确的技术构件规范化。与归一化的等位基因的比例显着不同的为1的标记表明，两个在同一个细胞的染色体产生的成绩单金额是不同的，这表明DNA与表型相关的变种基因表达的影响。应该用实验控制，以确认结果。

Watch this Scientific Journal Video about An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations at JoVE.com

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

遗传协会经常保持在功能级别上的原因不明。这种方法的目的是，通过分析细胞转录单核苷酸多态性杂合子基因表达，以评估的表型相关的遗传标记的影响。该技术可以精确测量，用MALDI - TOF质谱定量等位基因特异性引物延伸产品。

等位基因特异性基因表达检测，测试的遗传协会的功能基础

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

an-allele-specific-gene-expression-assay-to-test-functional-basis

Research

JoVE Journal

Biology

22.7K 次观看.  University of Oxford. 遗传协会经常保持在功能级别上的原因不明。这种方法的目的是，通过分析细胞转录单核苷酸多态性杂合子基因表达，以评估的表型相关的遗传标记的影响。该技术可以精确测量，用MALDI - TOF质谱定量等位基因特异性引物延伸产品。

视频: An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

光/暗转换测试小鼠

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

科研

生物学

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 ...

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

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 ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

一个反向遗传学方法测试胚胎发育过程中的功能冗余

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis 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 ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

药理和功能的遗传分析处理涡虫的再生日本三角涡虫</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

小规模的核提取物，基因表达机具功能分析

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...