Name: engineering artificial factors to specifically manipulate alternative splicing in human cells
Uploaded: 2017-04-26T13:00:00
Description: Watch this Scientific Journal Video about Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells at JoVE.com

这项工作是由美国国立卫生研究院资助R01-CA158283和国家自然科学基金委员会授予31400726到ZWYW由青年千人计划和中国国家自然科学基金（赠款31471235和81422038）的资助支持。 XY是由中国博士后科学基金会（2015M571612）资助。

通过重叠PCR 1. PUF支架与定制的构建RNA结合特异性<ol><li>设计一系列的包含在每个位置12（参照表1的引物序列和参见图1， 一种用于在RNA：PUF识别码）特异性识别不同的RNA核苷酸序列PUF的PCR引物。对于每个PUF重复，设计四个不同的引物识别每个位置的不同基地。 注意：这些引物将在一系列的四个回合的PCR反应中使?...

<ol>
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本报告详细描述了可以特异性操纵靶基因选择性剪接的人工剪接因子的设计和构建。该方法利用PUF重复的独特的RNA结合模式产生具有定制特异性的RNA结合支架。它可以用于激活或抑制拼接。 该协议中的关键步骤是生成定义了ESF特异性的重新编程的PUF结构域。已经开发并优化了PCR拼接方案，用于快速生成PUF支架。其成功的关键是将不同重叠模板的比例调整为1：1：1。每次循环?...

大多数的人类基因经历选择性剪接（AS），以产生多种同种型具有不同的活动，这大大增加了基因组1,2的编码的复杂性。 AS提供的主要机制来调节基因的功能，并且在不同的细胞和发育阶段3,4通过各种途径紧密调节。因为拼接错误调节是人类疾病5，6，7，8的常见原因，靶向剪接调节正在成为一种有吸引力的治疗路线。 根据剪接调节的简化模型，如由剪接调节顺 -elements（SRES）中前mRNA剪接增强子或替代外显子沉默发挥功能的主要控制。钍ESE的SRE具体招募各种反式作用蛋白因子促进或抑制剪接反应3,9（ 即剪接因子）。大多数反式作用剪接因子有单独的序列特异性RNA结合域认识到自己的目标和效应域控制剪接。最熟知的例子是丝氨酸/富含精氨酸的（SR）蛋白家族的含有N-末端的RNA识别基序（RRMS），其结合外显子剪接增强子，和C-末端RS结构域的成员，其中促进外显子内含10 。相反地，核蛋白A1通过RRM结构域结合至外显子剪接沉默子，并通过C末端的富含甘氨酸的结构域11抑制外显子包含。使用这种模块化配置，研究人员应该能够通过合并来设计人工剪接因子特异性RNA结合结构域（RBD）具有不同的短跑运动员激活或抑制剪接的结构域。 这种设计的关键是使用可编程RNA结合特异性识别给定靶标的RBD，其类似于TALE结构域的DNA结合模式。然而，大多数天然剪接因子含有RRM或K同源性（KH）结构域，其识别具有弱亲和力的短RNA元件，因此缺乏预测性RNA-蛋白质识别“代码” 12 。 PUF重复蛋白（ 即 PUF结构域）的RBD具有独特的RNA识别模式，允许重新设计PUF结构域以特异性识别不同的RNA靶标13,14 。典型的PUF结构域包含八个重复的三个α螺旋，每个重复在8-nt RNA靶标中识别单个碱基。第二个α-螺旋的某些位置的氨基酸侧链与t的Watson-Crick边缘形成特定的氢键他的RNA碱基决定了每个重复的RNA结合特异性（ 图1A ）。 PUF重复的RNA碱基识别代码令人惊讶地简单（ 图1A ），允许产生识别任何可能的8碱基组合的PUF结构域（由Wei和Wang 15综述）。 该模块化设计原则允许产生由定制的PUF域和剪接调制域（ 即 SR域或富含Gly域）组成的工程化拼接因子（ESF）。这些ESFs可以作为剪接活化剂或作为抑制剂来控制各种类型的剪接事件，并且已被证明可用作操纵与人类疾病相关的内源基因的剪接16,17 。例如，我们已经构建了PUF-Gly型ESFs来特异性地改变Bcl-x基因的剪接，将抗凋亡长的同种型（Bcl-xL）转化为促细胞凋亡的短异构体（Bcl-xS）。改变Bcl-x同种型的比例足以使几种癌细胞对多种抗癌化学药物16敏感，这表明这些人为因子可能作为潜在的治疗试剂。 除了用已知的剪接效应子结构域（ 例如， RS或富含Gly的结构域）控制剪接之外，工程化PUF因子也可用于检查新的剪接因子的活性。例如，使用这种方法，我们已经证明，当结合到不同的前mRNA区域18时，几种SR蛋白质的C-末端结构域可以激活或抑制剪接，RBM4的富含丙氨酸的基序可以抑制剪接19 ，并且DAZAP1的富含脯氨酸的基序可以增强剪接20,21/ sup&gt;。这些新功能域可用于构建其他类型的人为因子以微调拼接。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">High-fidelity DNA polymerase (Phusion High-Fidelity) with PCR buffer</td>
			<td style="width:155px;">New England Biolabs</td>
			<td style="width:103px;">M0530L</td>
			<td style="width:491px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA ligase (T4 DNA ligase)</td>
			<td>New England Biolabs</td>
			<td>M0202L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liposomal transfection reagent (Lipofectamine 2000)</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reduced serum medium (Opti-MEM)</td>
			<td>Gibco</td>
			<td>31985-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNA extraction buffer (TRIzol Reagent)</td>
			<td>ambion</td>
			<td>15596018</td>
			<td>TRIzol reagent includes phenol, which can cause burns. Wear gloves when handling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA (Bovine Serum Albumin)</td>
			<td>Sigma-Aldrich</td>
			<td>A7638-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS (1X)</td>
			<td>Life Technologies</td>
			<td>10010-031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperScript III reverse transcriptase</td>
			<td>Invitrogen</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Caspase-3 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9668</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PARP&#160;antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bcl-x antibody</td>
			<td>BD Bioscience</td>
			<td>610211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">beta-actin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>A5441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">alpha-tubulin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>T5168</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FLAG antibody</td>
			<td>Sigma-Aldrich</td>
			<td>F4042</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose membrane</td>
			<td>Amersham-Pharmacia</td>
			<td>RPN203D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECL Western Blotting detection reagents</td>
			<td>Invitrogen</td>
			<td>WP20005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy5-dCTP</td>
			<td>GE Healthcare</td>
			<td>PA55021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence-activated cell sorter</td>
			<td>BD Bioscience</td>
			<td>FACSCalibur&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td>GE Healthcare</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propidium iodide (PI)</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7638-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton-X100</td>
			<td>Promega</td>
			<td>H5142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-lysine&#160;</td>
			<td>Sigma</td>
			<td>P-4832</td>
			<td>Filter sterilize and store at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vector&#160; pWPXLd</td>
			<td>Addgene</td>
			<td>12258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vector pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vector psPAX2</td>
			<td>Addgene</td>
			<td>12260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I (RNase-free)</td>
			<td>New England Biolabs</td>
			<td>M0303S</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Oligo(dT)18 Primer</td>
			<td>Thermo Scientific</td>
			<td>SO131&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse secondary antibody (Anti-mouse IgG, HRP-linked Antibody)</td>
			<td>Cell Signaling Technology</td>
			<td>7076S</td>
			<td></td>
		</tr>
	</tbody>
</table>

engineering artificial factors to specifically manipulate alternative splicing in human cells

大多数真核RNA的处理是由RNA介导的结合的模块化配置，包括一个RNA识别模块，其特异性地结合前mRNA靶和效应结构域的蛋白质（限制性商业惯例）。以前，我们已经利用所述PUF结构域的独特RNA结合模式在人类Pumilio 1，以产生一个可编程的RNA结合的支架，将其用于设计各种人造限制性商业惯例来操纵RNA代谢。这里，详细的协议描述来构建专门设计用于调节靶基因的可变剪接工程剪接因子（ESFS）。该协议包括如何设计和构建定制的PUF支架用于特定RNA靶，如何通过融合设计者PUF结构域和效应子结构域，以及如何使用ESFS来操纵目标基因的剪接以构建ESF表达质粒。在该方法的代表性结果，我们还描述了常见的测定法的使用拼接记者，ESF的培养的人细胞中的应用，和剪接改变后续效应ESF活动。按照本报告中的详细协议，它可以设计并为不同类型的选择性剪接（AS）的规ESFS，提供了一个新的战略研究剪接调节和不同的剪接异构体的功能。此外，通过用设计PUF域融合不同的功能结构域，研究人员可以设计靶向特定RNA操纵RNA加工的各种步骤人为因素。

这份报告描述了ESFS和拼接记者的设计和施工的完整的协议。它还概述ESFS的操纵内源基因16的AS的进一步应用。为了说明的ESF介导的拼接变化的典型结果，我们使用的数据来自我们以前的工作作为一个例子。具有不同的功能结构域的ESFS可用于促进或抑制靶盒外显子（ 图1 D＆E）的夹杂物。 ESFS也可影响在报道?...

Watch this Scientific Journal Video about Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells at JoVE.com

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

该报告介绍了一种生物工程方法来设计和构建特异性调节在哺乳动物细胞中靶基因的剪接新颖人工剪接因子（ASFS）。该方法可以进一步扩展来设计各种人为因素来操纵RNA代谢的其它方面。

工程人为因素来特异性地操纵人细胞中的选择性剪接

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Research

JoVE Journal

Genetics

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

科研

遗传学

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

CRISPR/Cas9 诱导人多潜能干细胞内源蛋白标记的应用

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

基于细胞的 sineup 非编码 rna 检测, 可专门增强 mrna 的翻译能力

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

基于细菌选择和生长的盘状细胞基因工程

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

用于分离共转录RNA处理事件的人工RNA聚合酶II伸长复合物

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates

伦蒂病毒介导基因在低附着板中制备的人类伪性沉默

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

哺乳动物细胞中基于CRISPR的聚集基因屏幕

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...