Name: lentiviral mediated delivery of shrnas to hescs and npcs using low-cost cationic polymer polyethylenimine (pei)
Uploaded: 2022-05-24T15:00:00
Description: Watch this Scientific Journal Video about Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine PEI at JoVE.com

这项工作得到了阿拉伯联合酋长国大学（UAEU）的研究资助，赠款#31R170（扎耶德健康科学中心）和#12R010（UAEU-AUA赠款）的支持。我们感谢兰德尔·莫尔斯教授（纽约州奥尔巴尼沃兹沃思中心）帮助我们编辑手稿的风格和语法。
所有数据均可根据要求提供。

1. 使用 PEI 或脂质体酪胺 3000 试剂转染 HEK293T 细胞
<ol><li>在DMEM + 10%FBS + 1x青霉素/链霉素中在37°C下在具有5%CO2和21%O 2气氛的加湿培养箱中培养HEK293T细胞，直到它们在接种转染前90%汇合。使用相对较低的细胞传代数进行高滴度病毒生产（理想情况下小于P30）。</li>
	<li>在100mm组织培养板中的10mL完全生长培养基中接种4×106 个细胞，并在具有5%CO2和21 %O 2气?...

<ol>
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出于研究或临床目的对干细胞进行基因修饰的能力受到技术和对hESCs生物学的基本理解的限制。在小鼠ESC中显示出巨大潜力的技术，如脂质渗透和电穿孔，对hESCs来说效率不高，众所周知，hESCs很难通过常规方法进行基因传递20。这一概念不仅优化了现有技术，还开发了提高hESC转染效率的新方法。这些新发展的重点是高效和稳定的基因递送到hESC，同时在很长一段时间内保持对hESC?...

来自囊胚内细胞团的人胚胎干细胞（hESCs）是多能的，可以根据 体外 条件下的外部因素分化成不同的细胞类型1，2。为了充分利用hESC的潜力，必须为这些细胞提供快速可靠的基因递送方法。传统上，所使用的技术大致可分为两种类型：非病毒和病毒基因递送系统3，4。更常用的非病毒基因递送系统是脂质感染、电穿孔和细胞核感染。非病毒递送系统是有利的，因为插入突变较少，免疫原性总体上降低5，6。然而，这些方法导致转染效率低下和瞬时基因表达持续时间短，这是长期分化研究的主要限制7.与脂质感染相比，电穿孔可提高转染效率。然而，它导致超过50%的细胞死亡8，9，10。使用细胞核感染，可以通过结合脂质感染和电穿孔来提高细胞存活率和转染效率，但该方法需要细胞特异性缓冲液和专用设备，因此，对于放大应用来说，成本相当高11，12。
相比之下，病毒载体在转导后显示出更好的转染效率以及整体低细胞毒性。此外，递送的基因稳定表达，因此，这种方法非常适合长期研究13。将基因递送到hESC中最常用的病毒载体是慢病毒载体（LVS），使用高滴度病毒颗粒14，15可以提供超过80%的转导效率。脂质法转染和CaPO4 沉淀是瞬时转染HEK293T细胞或其衍生物以及具有基因转移载体的包装质粒以产生慢病毒颗粒16的最常用方法之一。虽然脂肪法感染具有良好的转染效率和低细胞毒性，但该技术受到其成本的阻碍，并且扩大规模以获得高滴度的慢病毒颗粒将非常昂贵。CaPO4 沉淀的转染效率与使用脂质法的转染效率相对相似。尽管CaPO4 沉淀具有成本效益，但在转染后会导致明显的细胞死亡，这使得标准化和避免批次间差异变得困难17.在这种情况下，开发一种具有高转染效率，低细胞毒性和成本效益的方法对于生产用于hESC的高滴度慢病毒颗粒至关重要。
聚乙基亚胺（PEI）是一种阳离子聚合物，可以高效率转染HEK293T细胞而没有太大的细胞毒性，并且与基于脂质感染的方法相比，成本可以忽略不计18。在这种情况下，PEI可用于通过使用各种技术从培养上清液中浓缩慢病毒颗粒来生产高滴度LVS的放大应用。本文介绍了使用 PEI 转染 HEK293T 细胞，以及通过蔗糖缓冲液进行超速离心的慢病毒载体浓缩。使用这种方法，我们定期获得远高于5 x 107 IU / mL的滴度，并且批次间差异很小。该方法简单，直接且具有成本效益，适用于将基因递送到hESC和hESCs衍生细胞的放大应用。

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			<td>Invitrogen</td>
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			<td>2118S</td>
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			<td>Gentle Cell Dissociation Reagent</td>
			<td>Stem Cell Technologies</td>
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			<td>HyClone Non Essential Amino Acids (NEAA) 100X Solution</td>
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			<td>Neuropan 2 Supplement 100x</td>
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			<td>Neuropan 27 Supplement 50x</td>
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			<td>Penicillin streptomycin SOL</td>
			<td>Invitrogen</td>
			<td>15140122</td>
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			<td>pLKO.1 TRC vector</td>
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			<td>Addgene</td>
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			<td>Polybrene infection reagent</td>
			<td>Sigma</td>
			<td>TR1003- G</td>
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			<td>Polyethylenimine, branched</td>
			<td>Sigma</td>
			<td>408727</td>
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			<td>psPAX2.0</td>
			<td>Addgene</td>
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			<td>Purmorphamine</td>
			<td>Tocris</td>
			<td>4551/10</td>
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			<td>Puromycin</td>
			<td>Invitrogen</td>
			<td>A1113802</td>
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			<td>ROCK inhibitor Y-27632 
			dihydrochloride&#160;</td>
			<td>Tocris</td>
			<td>1254</td>
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			<td>SB 431542</td>
			<td>Tocris</td>
			<td>1614/10</td>
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			<td>Trypsin .05% EDTA&#160;</td>
			<td>Invitrogen</td>
			<td>25300062</td>
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			<td>XAV 939</td>
			<td>Tocris</td>
			<td>3748/10</td>
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lentiviral mediated delivery of shrnas to hescs and npcs using low-cost cationic polymer polyethylenimine (pei)

目前的方案描述了使用慢病毒颗粒以高效率将短发夹RNA（shRNA）递送到人胚胎干细胞（hESCs）以及来自hESCs的神经祖细胞（NPC）。通过使用进入载体（携带shRNA）共转染HEK293T细胞以及使用低成本阳离子聚合物聚乙基亚胺（PEI）的包装质粒（pAX和pMD2.G）来生成慢病毒颗粒。使用超速离心浓缩病毒颗粒，这导致平均滴度高于5×107。hESCs和NPC都可以使用这些慢病毒颗粒高效感染，如嘌呤霉素的选择和在hESCs中的稳定表达以及NPC中的瞬时GFP表达所示。此外，蛋白质印迹分析显示，shRNA靶向基因的表达显著降低。此外，这些细胞保留了它们的多能性和分化潜力，正如它们随后分化成不同的CNS谱系所证明的那样。目前的协议涉及shRNA的交付;然而，相同的方法可用于cDNA的异位表达以进行过表达研究。

基因转移后，不可避免地需要高活性的hESC。尽管在hESCs电穿孔后努力并优化了方案以减少细胞死亡，但在电穿孔这些细胞后仍然观察到超过50%的细胞死亡，以及低转染效率20。慢病毒介导的基因转移不仅导致基因转移的高效率，而且在转导后显示出高水平的细胞活力。以下结果给出了两种方法：一种是使用GFP作为报告基因，在hESCs衍生的NPC中瞬时表达，另一种是使用嘌呤霉素稳定...

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Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine PEI

利用低成本的阳离子聚合物聚乙氨基亚胺（PEI），我们生产了慢病毒颗粒，用于H9人胚胎干细胞（hESCs）中shRNA的稳定表达，并高效瞬时转导H9衍生的神经祖细胞（NPC）。

慢病毒介导的shRNA递送到hESC和NPC，使用低成本阳离子聚合物聚乙亚胺（PEI）

The current protocol describes the use of lentiviral particles for the delivery of short hairpin RNAs (shRNAs) to both human embryonic stem cells (hESCs) ...

HEK293T细胞的转染必须在90%汇合度下进行。此外，使用0.45微米低蛋白结合过滤器进行慢病毒载体过滤和使用蔗糖垫进行超速离心也是必不可少的。首先在含有10%FBS和1X青霉素链霉素的DMEM培养基中，在37摄氏度的加湿培养箱中培养HEK293T细胞，在2%二氧化碳和21%氧气的气氛中，直到细胞达到90%汇合，然后接种进行转染。在100毫米组织培养板中，在10毫升完全生长培养基中将10至6个细胞接种4次，并在具有5%二氧化碳和21%氧气气氛的加湿组织培养箱中生长过夜。对于每次转染，按照文本手稿中所述的进入和包装质粒的比例，在1.5毫升离心管中，在一毫升无血清DMEM培养基中稀释每毫升质粒DNA储备品一毫克。涡旋管10秒，并在室温下以10， 000次G旋转30秒以收集。然后，向管中加入70微升每毫升聚合物聚乙烯亚胺或PEI储备溶液一毫克。再次，涡旋并短暂旋转管子。对于基于脂质番茄的转染，将载体稀释在500微升的无血清DMEM培养基中，该培养基含有试剂盒中提供的45微升试剂P。然后，使用500微升的相同介质稀释70微升试剂L。将两个试管在室温下孵育五分钟。然后，将两个管的内容物合并并在室温下孵育20分钟以允许复杂的形成。接下来，将一毫升DNA PEI或DNA脂质甲胺复合物滴加到含有细胞的板中，并在37摄氏度下在5%二氧化碳和21%氧气的气氛下孵育六小时。孵育后，用10毫升新鲜完全生长培养基替换培养基，并将板返回到培养箱中。转染两天后，收集含病毒培养基，并用10毫升新鲜完全生长培养基覆盖细胞。同样，在转染三天后，收集含病毒的培养基，并将其与在两天时间点收集的培养基相结合。将病毒上清液以2， 000次G离心10分钟，在四摄氏度下沉淀细胞碎片。使用0.45微米孔径低蛋白结合过滤器过滤上清液，并在4摄氏度下储存，直到准备好进行超速离心。用70%乙醇洗涤10分钟，对超速离心管的保温杯进行灭菌。风干，关闭，并将杯子置于四摄氏度。在含有慢病毒颗粒的过滤介质中加入36毫升到无菌超速离心管中。用在PBS中制备的四毫升无菌20%蔗糖溶液填充五毫升无菌条带，并将其分配到含有慢病毒载体或LVS的管的底部。确保蔗糖溶液不应与培养基混合，并在管子底部产生梯度。使用无血清DMEM培养基平衡所有超速离心管，将试管置于保温杯的冷超速离心管中，然后合上盖子。在4摄氏度下以125， 000次G旋转管两小时。旋转后，通过将管的内容物倒置在含有漂白剂的容器中而不干扰沉淀物来小心地丢弃上清液。标记颗粒（如果可见）。将超速离心管置于50毫升无菌管中，并在沉淀顶部加入200微升无菌DPBS。将管子置于四摄氏度下过夜。第二天，通过上下移液轻轻混合管的内容物，然后在台式离心机中以13， 000倍G旋转以沉淀任何碎屑。将上清液转移到新管中并等分试样病毒制剂。最后，将管子储存在零下80摄氏度。转染后，在HEK293T细胞中观察到丰富的GFP表达。病毒蛋白的表达导致HEK293T细胞的合胞体形成，其中单个细胞融合。感染后，在病毒感染的神经祖细胞中也观察到GFP的显着表达。低成本PEI和脂质菲卡明3000试剂之间的LVS滴度测量在病毒滴度上没有显示出任何显着差异。基于PLK 0.1的载体的病毒滴度如下所示。与非转导细胞相比，慢病毒转染细胞显示出超过80%的细胞活力，而没有细胞在治疗中存活下来。蛋白质印迹分析显示，L-2-羟基戊二酸脱氢酶在用空载体骨架转导的细胞中表达丰富。然而，在用携带短发夹RNA的载体转导的细胞中没有观察到表达。该技术的主要优点是使用低成本的阳离子聚合物聚乙烯亚胺通过使用入口和包装载体共转染HEK293T细胞来产生慢病毒颗粒。

Research

JoVE Journal

Biology

Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine (PEI)

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

科研

生物学

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

从MEFs基质胶我的MEFs传代的胚胎干细胞

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.
...

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

从MEFs基质胶2：到基质胶从MEFs分裂的胚胎干细胞

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

从MEFs基质胶3：传代的胚胎干细胞从基质胶到基质胶

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

长期沉默的1S Intersectin的在小鼠肺的特异性siRNA，通过阳离子脂质体重复交货。通过电子显微镜的击倒效果评价

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

采用共培养，以检测化学介导的种间相互作用

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Myospheres的产生从人类胚胎干细胞通过表观遗传修饰

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

啮齿动物附睾精子磷酸分析

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...