这项工作是由美国国立卫生研究院在奖数R01HG006785美国国家人类基因组研究所的支持。

1序列的设计与合成<ol><li>开始MPRA与序列的设计和合成，来测定调节活性。用于与pMPRA矢量系列的相容性，使用以下模板设计每个序列:5&#39;-ACTGGCCGCTTCACTG- 变种 -GGTACCTCTAGA- 标签 -AGATCGGAAGAGCGTCG-3&#39; 其中 var表示序列待测定和标记表示一个或多个识别标记。 </li><li>两个可变区（var和标签 ）通过一对的KpnI（GGTACC）和XbaI（TCTAGA）限制性位点的分离，以促进它们之间的报道基因片段的定向连接。此外，两个不同的SfiI（GGCCNNNNNGGCC）网站将通过PCR被添加以允许定向连接到pMPRA骨干。可变区必须不包含任何这些限制性位点的附加拷贝。如果需要的话，其中的一个限制性内切酶的一个或多个可更换，只要更换1）能够进行高效率的切削接近的DNA分子的末端，以及2）没有在载体序列别处切割。见讨论有关其他详细信息。 </li><li>获得来自商业供应商如表1所列的所需的引物。 </li></ol><table border="0" cellpadding="0" cellspacing="0" width="381"><tbody><tr><td> MPRA_SfiI_F </td><td> GCTAAGGGCCTAACTGGCCGCTTCACTG </td></tr><tr><td> MPRA_SfiI_R </td><td> GTTTAAGGCCTCCGTGGCCGACGCTCTTC </td></tr><tr><td> TAGseq_P1 </td><td> AATGATACGGCGACCACCGAGATCTACACT CTTTCCCTACACGACGCTCTTCCGATCT </td></tr><tr><td> TAGseq_P2 </td><td> CAAGCAGAAGACGGCATACGAGAT [指数] GT GACTGGAGTTCAGACGTGTGCTCTTCCGATCTCGAGG </td></tr></tbody></table> 表1引物序列[指数]表示用于多路测序6至8个核苷酸指数序列。获得至少8 TagSeq-P2引物与不同的索引。所有的引物应通过HPLC或PAGE纯化。 <ol start="4"><li>寡核苷酸文库可从商业供应商或核心设施来获得，或者通过使用一个可编程微阵列为基础的寡核苷酸合成仪所描述的制造商产生。悬浮OLS的产品在100μlTE缓冲液0.1。 </li><li>运行在10％TBE-尿素变性聚丙烯酰胺凝胶中的寡核苷酸文库。负载每通道和染色约1皮摩尔与单链DNA敏感的荧光染色，以评估其质量（ 图2A）。 </li><li>如果对应于全长寡核苷酸的离散频带可以被可视化（ 图2A，泳道1和2），切下相应的丙烯酰胺凝胶切片和洗脱的寡核苷酸到100微升的TE 0.1过夜，在室温下摇动。否则，继续使用原始OLS停牌。 </li><li>放大和使用的乳液PCR 9添加的SfiI尾巴寡核苷酸文库</<html> SUP&gt;。 </li><li>如表2中描述的建立50μl的PCR反应混合物。从Micellula DNA乳剂300μl的油和表面活性剂的混合物和纯化试剂盒和涡流在4℃下再结合5分钟。 </li></ol><table border="0" cellpadding="0" cellspacing="0" width="368"><tbody><tr><td>试剂</td><td> 1X量（μl） </td></tr><tr><td> Herculase II融合DNA聚合酶</td><td> 0.5 </td></tr><tr><td> 5X Herculase II反应缓冲液</td><td> 10 </td></tr><tr><td>的dNTP（各10毫米） </td><td> 1.25 </td></tr><tr><td> BSA（20毫克/毫升） </td><td> 1.25 </td></tr><tr><td>底漆MPRA_SfiI_F（25微米） </td><td> 0.25 </td></tr><tr><td>底漆MPRA_SfiI_R（25微米） </td><td> 0.25 </td></tr><tr><td> OLS模板（1-10渺摩尔） </td><td>变化</td></tr><tr><td>无核酸酶的水</td><td> 50 </td></tr></tbody></table>ve_content"&gt; 表2乳液PCR反应混合物（水相）。 <ol start="9"><li>使用给出了扩增产物的最大产量没有任何瑕疵的出现浓度（见1.12）。 </li><li>分配所得乳液进PCR管中，并在95℃下进行20个PCR循环（2分钟，20×[20秒，在95℃，20秒，55℃，30秒，72℃]，3分钟，在95 ℃）。 </li><li>池和破每350微升的乳液PCR反应通过加入1 ml异丁醇，并简要涡旋，然后纯化，使用DNA纯化柱的扩增产物。 </li><li>运行在2％或4％的琼脂糖凝胶的等分试样，以验证无伪影的扩增（ 图2B）。 </li></ol><img alt="图2" src="/files/ftp_upload/51719/51719fig2highres.jpg" /> 图2制备寡核苷酸合成库。一） </s仲&gt;三种不同原料合成的寡核苷酸库法（OLS），在变性10％TBE-尿素聚丙烯酰胺凝胶运行。对应全长寡核苷酸（*）频段可以可视化，并从图书馆切除1和2图书馆3包含与页净化干扰污染物。如果是这种情况，直接进行相同的寡核苷酸库运行的打开和乳液PCR扩增的PCR扩增。 乙）产品在琼脂糖凝胶上。复杂的寡核苷酸文库PCR扩增经常造成嵌合产品，可能会出现更高和更低的频段等文物。乳液PCR技术可以最大限度地减少这些文物。 2，图书馆建设<ol><li>制备的线性化质粒骨架消化载体pMPRA1用SfiI位在50℃下搅拌2小时，在1％的琼脂糖凝胶电泳，切下的骨干频带（在这种情况下，2.5 kb的），并用凝胶纯化旋转柱纯化它。 </li><li>摘要从步骤1.4用SfiI位 ，在50℃下进行2小时的乳液PCR产物和纯化使用DNA纯化柱。 </li><li>结扎的子标签库成线性载体骨架，建立含有100毫微克的消化PCR产物，50毫微克的线性化载体骨架和T4 DNA连接酶反应。过夜孵育在16℃，然后加热，在65℃下进行20分钟以灭活连接酶。 </li><li>转化大肠杆菌大肠杆菌用连接反应。以保存库的复杂性，目标以外还有在设计库不同的启动子，标记的组合，以获得至少10倍以上的CFU。 </li><li>当标记的总数为约10万，则目标菌落通常可以通过进行每库6-8平行变换来实现的。 </li><li>对于每个转换，结合50微升电感受态大肠杆菌大肠杆菌细胞用2微升连接反应混合物在冰上。变换了电，恢复细胞800μLSOC培养基在37℃下振荡1小时，然后从并行变换相结合的细胞。 </li><li>评估转化效率，板连续稀释液从在LB琼脂平板上以50〜100微克/毫升羧苄青霉素的回收单元的alinquot孵育过夜，在37℃。估计总菌落为（观察到的CFU）×（稀释倍数）×（V - V）/ V，其中V是回收的细胞和v的总体积是采取的系列稀释的等分试样的体积。 </li><li>同时，使用回收的细胞的其余接种200毫升LB补充有100微克/毫升羧苄青霉素。在摇床培养箱中生长的细胞在37℃下过夜，然后分离出质粒DNA，按照标准方法。 </li><li>消化用SfiI位的分离的质粒文库的等分试样在50℃下搅拌2小时，并运行在1％琼脂糖凝胶上，以证实插入片段的存在。 </li><li>通过线性C的质粒库用KpnI和XbaI位启动子变异与标签之间utting。为了最大限度地提高消化效率，执行串行消化: </li><li>首先，摘要用KpnI，37℃1小时，纯化使用磁珠。第二，消化用XbaI并加入1 U虾碱性磷酸酶，在37℃下搅拌2小时，热失活，在65℃下进行5分钟，然后纯化使用磁珠。 </li><li>运行在1％琼脂糖凝胶的等分试样，以证实完全线性化。如果未酶切的质粒是可见的，线性化的片段应该被凝胶纯化。 </li><li>以产生适合于转染到哺乳动物细胞中的MPRA库，结扎的ORF用KpnI / XbaI位兼容的末端插入线性化中间库。 </li><li>要准备从pMPRAdonor1兼容luc2的ORF片段，消化该质粒用KpnI和XbaI，37℃1小时，并运行在1％琼脂糖凝胶。消费税的ORF片段（1.7 K表在这种情况下，B）和纯化使用凝胶净化离心柱。 </li><li>克隆的ORF片段克隆到线性化的中间库与步骤2.3-2.8中描述。 </li><li>消化MPRA文库用KpnI的等分试样（相当于1-2微克），在37℃下反应1小时，并运行在1％的琼脂糖凝胶。如果消化库运行为单一条带，进入第3节。如果需要额外的频段观察（通常对应于中间结构的结转），该库可进一步纯化如下: </li><li>消化3-5微克用KpnI污染库在37℃下1小时，在0.8％琼脂糖凝胶上，在4℃下运行过夜。 </li><li>切下正确的库带，使用凝胶纯化旋转柱纯化DNA，并使用高浓度T4 DNA连接酶进行1小时进行自身连接，在37℃。然后重复改造和最后文库DNA分离的步骤2.8所述。 </li></ol>把transfection，扰动和RNA分离<ol><li>对于每个独立的转染，培养的细胞所需要的数目（如由MPRA库的复杂性来确定，见讨论）在适当的培养基中。例如，培养的HEK293T / 17的细胞在DMEM补充有10％FBS和L-谷氨酰胺/青霉素/链霉素。培养的细胞为每个库和实验条件至少有两个独立的转染。 </li><li>转染培养的细胞以MPRA质粒。转染的方法和条件，必须为每个信元类型被优化。对于每一个转染的样品，保留了质粒DNA的匹配对照的等分（50-100毫微克）。 </li><li>例如，转染0.5×10 7个HEK293T /生长17细胞以〜于10cm的培养皿中含10微克质粒DNA在1ml的Opti-MEM I低血清培养基用加入30μl的Lipofectamine LTX和10μl加试剂50％汇合。 5小时后取出转染混合物，并允许细胞恢复24-48小时。 </li><li>任选地，进行激活在设计库上下文或信号相关的调控序列所需的任何扰动。 </li><li>收获细胞，并使用使用其制造商的指示标准寡聚（dT）纤维素柱或磁珠分离出的poly（A）+ mRNA的表达。 </li><li>确保该列或珠粒的最大结合容量超过mRNA的从收获的细胞预期的总量。例如，从3.3节的预期收益率大约为0.5-2.5微克表达。 </li></ol> 4，标签序列<ol><li>消除的载体DNA的交叉污染，从被转染的细胞裂解物，对待每20微升的mRNA样品与1μl的涡轮DNA酶（2U）和2.3微升10倍的Turbo DNA酶缓冲液中于37℃下反应1小时，加入2.4微升的Turbo DNA酶失活的试剂在RT下5分钟，搅拌，离心分离机以10,000 g下90秒，然后将溶液转移到新的管中。 </li><li>验证<html>纯度在琼脂糖凝胶上进行PCR作为60-100纳克的mRNA样品的4.6节中描述，然后运行该产品。如果特异性扩增是可见的（0.25 KB，如果使用pMPRA1与luc2），柱净化处理后的mRNA，然后重复DNase处理。 </li><li>要生成标签，序列测序文库，转换报告mRNA合成cDNA，经PCR测序添加适配器。 </li><li>设置的mRNA / RT引物的混合物，如表3所述。孵育在65℃下5分钟，然后放置在冰上。并联，建立cDNA合成反应中，如表4所述。 </li></ol><table border="0" cellpadding="0" cellspacing="0" width="428"><tbody><tr><td>试剂</td><td> 1X量（μl） </td></tr><tr><td>基因样本（400-700纳克总） </td><td> 8 </td></tr><tr><td>寡0dT（50微米） </td><td> 1 </td></tr><tr><td>的dNTP（各10毫米） </td><td> 1 </td></tr></tbody></table>ove_content"&gt; 表3的RNA /反转录引物混合cDNA合成。 <table border="0" cellpadding="0" cellspacing="0" width="428"><tbody><tr><td>试剂</td><td> 1X量（μl） </td></tr><tr><td> 10倍的Superscript III RT缓冲液</td><td> 2 </td></tr><tr><td> 氯化镁 （25毫米） </td><td> 4 </td></tr><tr><td> DTT（0.1M） </td><td> 2 </td></tr><tr><td> RNaseOut（40 U /μL） </td><td> 1 </td></tr><tr><td>的Superscript III（200 U /μL） </td><td> 1 </td></tr></tbody></table> 表4的cDNA合成反应混合物中。 <ol start="5"><li>轻轻地结合cDNA合成的混合基因/ RT引物混合。孵育在50℃下50分钟，85℃下进行5分钟，然后放置在冰上。最后，孵育带U核糖核酸酶的2小时，在37℃下进行20分钟。 </li><li>设置PCR反应，如表5中所述的4-6微升cDNA的反应混合物或50毫微克报告质粒作为模板。然后进行PCR扩增（95℃，2分钟，26×[95℃30秒，55℃30秒，72℃30秒]，72℃，3分钟）。 </li></ol><table border="0" cellpadding="0" cellspacing="0" width="428"><tbody><tr><td>试剂</td><td> 1X量（μl） </td></tr><tr><td> 2个PfuUltra II热启动PCR反应混合液</td><td> 25 </td></tr><tr><td>底漆TagSeq_P1（25微米） </td><td> 0.5 </td></tr><tr><td>底漆TagSeq_P2（25微米） </td><td> 0.5 </td></tr><tr><td>模板（mRNA和cDNA的混合物或质粒DNA） </td><td>变化</td></tr><tr><td>无核酸酶的水</td><td> 50 </td></tr></tbody></table> 表5变量-SEQ PCR反应混合物。 <ol start="7"><li>如果使用通过2％琼脂糖凝胶上运行PCR产物，对应于该标签的序列文库的扩增子的附加频带（0.25 kb的pMPRA1与luc2）和纯化这些使用凝胶净化离心柱。 </li><li>游泳池，变性，直接与Illumina的测序仪测序纯化标签序列扩增。 </li><li>过滤低质量的撤离所有，一个读）包含了PHRED质量的一个或多个位置排序的标签中得分低于30或b）未在标签设计完全匹配。算上其余每个标签出现在每个库的次数。归一化标签计数到TPM（每百万测序标记标签），然后再计算的mRNA衍生的标签计数超过质粒衍生的标签数为每对测序文库的比率。如果有多个不同的标签被链接到每个序列变异体，用他们的平均比例为下游分析。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

MPRA is a flexible and powerful tool for dissection of sequence-activity relationships in gene regulatory elements. The success of MPRA experiments depend on at least three factors: 1) careful design of the sequence library, 2) minimization of artifacts during amplification and cloning, and 3) high transfection efficiency. 
The possible lengths of the variable regions in the reporter constructs are largely determined by the synthesis or cloning technology used. Standard OLS is generally limited to about 200 nt, but this protocol is compatible with inserts up to at least 1,000 nt. Note that variable regions that are highly repetitive or contain strong secondary structures may end up underrepresented due to PCR and cloning biases. The length of the tags that identify each of the variable regions should be 10-20 nt and the collection of tags should ideally be designed such there are at least two nucleotide differences between any pair. Tags that contain the seed sequences of known microRNA or other factors that might influence mRNA stability should also be avoided when possible.
A key parameter in the design of MPRA experiments is the total number of distinct reporter constructs to be included in the library (the design complexity, denoted CD). In practice, CD is limited by the number of cultured cells that can be transfected. As a rule of thumb, the total number of transfected cells should be at least 50-100 times greater than CD. For example, if 20 million cells can be transfected with a transfection efficiency of 50%, then CD should be no more than ~200,000. Note that CD is equal to the number of distinct regulatory sequence variants multiplied by the number of distinct tags per sequence. The more distinct tags are linked to each regulatory sequence, the more accurate the estimate of the activity of that sequence can be made (because measurements from distinct tags can be averaged), but the fewer distinct variants can be assayed in one experiment. The optimal choice depends on the experimental design. In a simple &#8220;promoter bashing&#8221; experiment, where a mathematical model will be fitted to the aggregated measurements, a single tag per variant is usually sufficient. In a screen for single-nucleotide polymorphisms that cause changes in regulatory activities, it may be necessary to use 20 or more tags per allele to obtain statistically robust results, because comparing each pair of alleles requires a separate hypothesis test. 
If the sequences to be assayed are not expected to contain transcription start sites, a constant promoter can also be added in the same fragment. For example, pMPRAdonor2 (Addgene ID 49353) includes a minimal TATA-box promoter that is useful when the upstream variable region is expected to have significant enhancer activity, while pMPRAdonor3 (Addgene ID 49354) includes a modified, strong SV40 viral promoter that is useful when the variable region is expected to contain silencer activity or other negative regulatory elements. 
Raw OLS products often contain a significant fraction of truncated oligonucleotides. These may interfere with accurate PCR amplification of the designed sequences, particularly when there is significant homology between them. Using PAGE purification to remove truncated synthesis products and emulsion PCR to minimize amplification artifacts are effective techniques for ensuring high library quality. If either step is impractical, it is imperative to minimize the number of PCR cycles used at each amplification step. Selection and expansion of the cloned library in liquid culture is generally sufficient to maintain the design complexity, but if recombination-prone vectors are to be used or significant representation bias is observed, the recovered cells can instead be plated directly onto large LB agar plates, expanded as individual colonies and then scraped off for DNA isolation. It is also important to consider the potential impact of synthesis errors, which are typically found at a rate of 1:100-500 in OLS. Full-length sequencing of the reporter constructs prior to transfection is recommended to identify and correct for such errors.
It is not necessary to introduce reporter constructs into every cell in the transfected culture, but transfection efficiencies below ~50% may lead to poor signal to noise ratios. It is advisable to optimize transfection conditions prior to performing MPRA experiments in a new cell type. When working with hard-to-transfect cell types, MPRA signals can be boosted by pre-selecting transfected cells. The pMPRA vector series includes variants that constitutively express a truncated cell surface marker that can be used to physically enrich for transfected cells prior to RNA isolation (for example, Addgene IDs 49350 and 49351).

A correction to the author list has been made for article <a href="http://www.jove.com/video/51719">Massively Parallel Reporter Assays in Cultured Mammalian Cells</a>.

Erratum: Massively Parallel Reporter Assays in Cultured Mammalian Cells

大规模并行报告基因分析（MPRA）允许数以千计的转录调节活性的复用测量到几十万的DNA序列1-7的。在其最常见的实施方式中，复用通过连接每个感兴趣的序列，其中包含一个开放读框下游的一个识别序列标签的合成报道基因来实现（ORF; 图1）。转染后，RNA分离和报道基因转录物的3&#39;末端的深度测序，耦合的序列的相对活动，可以从它们的识别标记的相对丰度来推断。 <img alt="图1" src="/files/ftp_upload/51719/51719fig1highres.jpg" /> 图1概述MPRA的。MPRA记者库构建体是由耦合假定调控序列来合成构造其次是一个标识序列标签的报告基因，它由一个"惰性"ORF（如GFP或萤光素酶）。该文库转染集体到培养的细胞群和转录的mRNA记者随后恢复。深度测序用于计算出现在记者的mRNA和转染的质粒中的每一标签的数量。 mRNA的比率计算过的质粒计数可以用来推断相应的调节序列的活性。适于与从尼科夫权限等 2。 MPRA可适应各种实验设计，包括个体基因的调节元件的1）完整的突变，2）扫描横跨感兴趣的基因座的新的调节元件，3）测试中的一组假定的自然遗传变异的影响启动子，增强子或沉默，而合成的调控元件4）半理性设计。李可以采用多种方法，包括寡核苷酸文库合成法（OLS）的可编程芯片2,3,6,7，组装简并寡核苷酸1,4，组合结扎8和基因组DNA 5碎裂产生的序列变异中图书馆。 本协议描述的建设启动的库变体使用OLS和pMPRA1和pMPRAdonor1向量（Addgene识别码49349和49352，分别http://www.addgene.org），这个库的瞬时转染到培养的哺乳动物细胞和随后的定量通过它们的相关标签（TAG-SEQ）深度测序的启动子活性。早期版本的这个协议的报道，梅尔尼科夫等自然生物技术30，271-277（2012）和Kheradpour 等基因组研究23，800-811（2013）的研究中使用。

<table><tbody><tr><td>Oligonucleotide library synthesis</td><td>Agilent, CustomArray, or other OLS vendors</td><td>custom</td><td>If using OLS construction method</td></tr><tr><td>pMPRA1</td><td>Addgene</td><td>49349</td><td>MPRA plasmid backbone</td></tr><tr><td>pMPRAdonor1</td><td>Addgene</td><td>49352</td><td>luc2 ORF donor plasmid</td></tr><tr><td>TE 0.1 Buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0)</td><td>Generic</td><td>n/a</td><td>OLS buffer</td></tr><tr><td>Novex TBE-Urea Gels, 10%</td><td>Life Technologies</td><td>EC6875BOX</td><td>PAGE purification of OLS products</td></tr><tr><td>Novex TBA-Urea Sample Buffer</td><td>Life Technologies</td><td>LC6876</td><td>PAGE purification of OLS products</td></tr><tr><td>SYBR Gold Nucleic Acid Gel Stain</td><td>Life Technologies</td><td>S-11494</td><td>PAGE purification of OLS products</td></tr><tr><td>Micellula DNA Emulsion &amp;amp; Purification Kit</td><td>EURx/CHIMERx</td><td>3600-01</td><td>Library amplification by emulsion PCR</td></tr><tr><td>Herculase II Fusion DNA Polymerase</td><td>Agilent</td><td>600675</td><td>Polymerase for emulsion PCR</td></tr><tr><td>SfiI</td><td>New England Biolabs</td><td>R0123S</td><td>Library cloning with pMPRA vectors</td></tr><tr><td>KpnI-HF</td><td>New England Biolabs</td><td>R3142S</td><td>Library cloning with pMPRA vectors</td></tr><tr><td>XbaI</td><td>New England Biolabs</td><td>R0145S</td><td>Library cloning with pMPRA vectors</td></tr><tr><td>T4 DNA Ligase (2,000,000 units/ml)</td><td>New England Biolabs</td><td>M0202T</td><td>Library cloning with pMPRA vectors</td></tr><tr><td>One Shot TOP10 Electrocomp &lt;em&gt;E. coli&lt;/em&gt;</td><td>Life Technologies</td><td>C4040-50</td><td>Library cloning with pMPRA vectors</td></tr><tr><td>LB agar and liquid media with carbenicllin</td><td>Generic</td><td>n/a</td><td>Growth media for cloning</td></tr><tr><td>E-Gel EX Gels 1%</td><td>Life Technologies</td><td>G4010-01</td><td>Library verification and purification</td></tr><tr><td>E-Gel EX Gels, 2%</td><td>Life Technologies</td><td>G4010-02</td><td>Library verification and purification</td></tr><tr><td>MinElute Gel Extraction Kit</td><td>Qiagen</td><td>28604</td><td>Library and backbone purification</td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>Qiagen</td><td>12362</td><td>Library DNA isolation</td></tr><tr><td>Cell culture media</td><td>n/a</td><td>n/a</td><td>Experiment-specific</td></tr><tr><td>Transfection reagents</td><td>n/a</td><td>n/a</td><td>Experiment-specific</td></tr><tr><td>MicroPoly(A)Purist Kit</td><td>Life Technologies</td><td>AM1919</td><td>mRNA isolation</td></tr><tr><td>TURBO DNA-free Kit</td><td>Life Technologies</td><td>AM1907</td><td>Plasmid DNA removal</td></tr><tr><td>SuperScript III First-Strand Synthesis System</td><td>Life Technologies</td><td>18080-051</td><td>cDNA synthesis</td></tr><tr><td>PfuUltra II Hotstart PCR Master Mix</td><td>Agilent</td><td>600850</td><td>Polymerase for Tag-Seq PCR</td></tr><tr><td>Primers (see text)</td><td>IDT</td><td>custom</td><td>PAGE purify Tag-Seq primers</td></tr></tbody></table>

massively parallel reporter assays in cultured mammalian cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

MPRA利于高分辨率的转录调控元件的序列 - 活性关系的定量解剖。一个成功的MPRA实验通常会产生高度可重复的测量结果，为广大的转库（ 图3A）序列。如果重复性差被观察（ 图3B），这是指示记者mRNAs的回收的RNA样本中的浓度太低，由于是1）低的检测序列中的绝对活性，或2）低的转染效率。 图4显示了通过测定生成的具有代表性的"信息足迹"1,2〜人类IFNB基因在HEK293细胞有或没有接触到仙台病毒上游的145 bp的序列37000的随机变量。启动子的TATA盒和已知的近端增强10可以明确认定为信息丰富REGI附件中的病毒依赖性。 <img alt="图3" src="/files/ftp_upload/51719/51719fig3highres.jpg" /> 图3标签序列的重现性。散点图显示高（A）和低电平（B）的再现性，从 ​​两个独立的复制次转染标记-SEQ数据的例子。后者图示出许多异常的标记具有高的mRNA计数仅在一个在两个重复的。这样的工件通常表示记者mRNA的浓度太低，定量PCR扩增，或者是由于该报道构建中低绝对值的活动，或低的转染效率。 <img alt="图4" src="/files/ftp_upload/51719/51719fig4highres.jpg" /> 人类IFNB转录起始位点和近端增强图4，信息足迹。 </str翁&gt;上游IFNB人类基因约有37,000随机的145个核苷酸（nt）的区域变异采用MPRA在HEK293细胞（A）和无（二）接触到仙台病毒检测。蓝色条显示记者输出和核苷酸在每个位置之间的互信息。近端增强子和TATA盒中脱颖而出，作为对病毒感染的高信息含量的区域。

Watch this Scientific Journal Video about Massively Parallel Reporter Assays in Cultured Mammalian Cells at JoVE.com

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

在培养的哺乳动物细胞大规模并行报告基因分析

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

massively-parallel-reporter-assays-in-cultured-mammalian-cells

Research

JoVE Journal

Biology

21.5K 次观看.  Broad Institute. The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

视频: Massively Parallel Reporter Assays in Cultured Mammalian Cells

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

一种多功能自动化平台微尺度细胞刺激实验

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

科研

生物工程

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

化学

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

医学

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

免疫与感染

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

一种报告基因检测法，用于分析哺乳动物细胞中内旋微RNA成熟

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves ...

癌症研究

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

在Live哺乳动物细胞中质粒复制多代荧光显微镜监测

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

生物学

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new therapeutics. Mas related G-protein receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, directs the general immune response. Numerous ligands for MRGPRX2 have been identified and include endogenous peptides like PAMPs, defensins, LL-37 and other protein fragments (i.e., degraded albumin). Further identification of MRGPRX2 specific ligands requires the screening of a large number of peptides (i.e., peptide library); however, mast cells are difficult and expensive to maintain in vitro and, therefore, not economical to use for screening large numbers of molecules. The present paper demonstrates a method to design, develop, and screen a library of small peptide molecules using MRGPRX2 expressing HEK cells. This cell line is relatively easy and inexpensive to maintain and can be used for in vitro high-throughput analysis. A calcium sensitive Fura-2 fluorescent dye to mark intracellular calcium flux upon activation was used to monitor the activation. The ratio of fluorescence intensity of Fura-2 at 510 nm against excitation wavelengths of 340 and 380 nm was used to calculate calcium concentration. The peptide library used to verify this system was based on the endogenous proadrenomedullin N-terminal 12 (PAMP-12) secretagogue, which is known to bind MRGPRX2 with high specificity and affinity. Subsequent peptides were generated through amino acid truncation and alanine scanning techniques applied to PAMP-12. The method described here is simple and inexpensive yet robust for screening a large library of compounds to identify binding domains and other important parameters that play an important role in receptor activation.

Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors.

使用工程 HEK 细胞筛选激活 MRGPRX2 的肽

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...