Name: small rna transfection in primary human th17 cells by next generation electroporation
Uploaded: 2017-04-13T15:00:00.000+00:00
Duration: 10 min 15 s
Description: Watch this Scientific Journal Video about Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation at JoVE.com

This work was supported by the US National Institutes of Health grants (R01HL109102, P01HL107202, U19CA179512, F31HL131361), a Leukemia &amp; Lymphoma Society scholar award (K.M.A.), and the National Institute of General Medical Sciences (NIGMS) Medical Scientist Training Program (Grant #T32GM007618) (M.M.).

该协议坚持加州大学旧金山分校的人类研究伦理指导原则。 1. T细胞培养，CD4 + T细胞的分离，和Th17极化的制备<ol><li> 在第0天，涂层6孔，每个抗人CD3（2微克/ mL）和抗人CD28的孔1.5ml组织培养板（4微克/毫升）的PBS与钙和镁为至少2小时37℃。 <ol><li>可替代地，涂层在4℃下过夜平板。裹在板封口膜。 </li></ol></li><li>制备脐带血单核细胞（CBMCs）通过根据制造商说明书密度梯度离心。 注意:仔细工作，确保适当的个人防护装备（PPE）处理人血的时候以避免暴露于血源性病原体的风险已磨损。 </li><li> 一旦单核细胞已被分离和洗涤，通过负塞莱执行人CD4 + T细胞分离ction使用市售的人CD4 + T细胞的试剂盒。 注意:如果有后单核细胞的分离红血细胞污染，任选的红血细胞裂解可以先于CD4 + T细胞分离的步骤中进行。重悬的单核细胞在1mL隔离缓冲液（2％FBS的PBS中）的。加入5毫升的1X溶胞液。孵育在室温下15分钟。然后在300×g下在4℃下加入5毫升分离缓冲液和离心机5分钟以沉淀细胞。 <ol><li>重悬的单核细胞在隔离缓冲器一个的50×10 6密度为每500μL在一个新的5mL的试管中。 </li><li>加入100μLFBS和100μL抗体混合物每管，然后在4℃下孵育各管20分钟定轨振荡器上充分混合的。 </li><li>在温育后，加入隔离缓冲器3-4 mL至洗涤细胞。离心将细胞在300×g离心8分钟，在4℃以沉淀细胞。小心吸苏佩rnatant。 </li><li>在这个旋转，预洗磁珠。转移的磁珠的所需量进新管中（在1中使用:1与细胞），并添加隔离缓冲器的等体积的洗涤珠。拌匀使用1种毫升微量，然后将管在磁体至少1分钟。小心吸出上清液。重悬在相同体积的初始前的洗涤传送的磁珠。 </li><li>将细胞重悬于每个管与隔离缓冲器500μL，并添加预洗涤磁珠500μL。 </li><li>孵育在室温下（18℃至25℃）的定轨摇床上以15分钟的磁珠的细胞拌匀。 </li><li>孵育后，彻底吸管使用1毫升微量的至少10倍的细胞。然后添加隔离缓冲器的3-4毫升，并放置各管在磁体2分钟。 </li><li>小心转移是在带负选择的CD4 + T细胞上清至新管。 </li></ol></li><li>一旦CD4 + T细胞分离完成后，计数与血球细胞和保持细胞在冰上。 </li><li>用PBS洗涤抗体包被的平板两次。然后1.5 Th17的偏振媒体的2倍的溶液混合添加到每个孔中:抗人IFNγ（20微克/毫升），抗人IL-4（20μg/ mL的），人TGFβ（10ng / mL的），人IL-1β（40毫微克/毫升），人IL-23（40毫微克/毫升），人IL-6（50毫微克/毫升）的所有稀释在无血清基础培养基（补充有2mM L-谷氨酰胺，100 U / ml的青霉素，100μg/ mL链霉素，10mM的HEPES，1mM丙酮酸钠和100μM2-巯基乙醇）。 注:转染和RNA干扰确实在含血清媒体工作。使用无血清培养基与该协议的目的是为了实现更好的IL-17A的生产。 </li><li> 离心机人类CD4 + T细胞在500×g离心5分钟，在4℃以沉淀细胞。小心吸supernat蚂蚁。然后重悬在无血清基础培养基的细胞和板上的细胞以3×10 6个的密度在每孔，使得最终体积为每孔3 mL和偏振细胞因子现在正处于1X终浓度1.5毫升。 <ol><li>将平板在5％CO 2，37℃的培养箱中两天。 </li></ol></li></ol> 2. 体外的电穿孔极化人Th17细胞<ol><li> 第2天，外套48孔每抗人CD3（2微克/ mL）和抗人CD28的井250μL的组织培养板（4微克/毫升）的PBS与钙和镁为至少2小时37℃。 <ol><li>可替代地，涂层在4℃下过夜平板。裹在板封口膜。 </li></ol></li><li>准备小RNA转染。对于每次转染，等分试样1 5μM储备溶液的siRNA到1.5mL微量离心管中的μL。包括适当的化学匹配的小RNA对照醇。置于冰上所有的管子。 </li><li>用PBS洗涤抗体包被的平板两次。然后添加500μL的1X的Th17极化介质到每个孔中:抗人IFNγ（10微克/毫升），抗人IL-4（10μg/ mL的），人TGFβ（5毫微克/毫升），人IL- 1β（20ng / mL的），人IL-23（20ng / mL的），人IL-6（25毫微克/毫升）的所有稀释在无血清基础培养基（补充有2mM L-谷氨酰胺，100U / mL青霉素，100微克/毫升链霉素，10mM的HEPES，1mM丙酮酸钠和100μM2-巯基乙醇）。 </li><li>制备的培养板中并转染试剂后，重悬以1种毫升微量，轻轻移液，但要确保所有的细胞从孔的底部分离的细胞。池中的细胞进入一个锥形管和离心机在500×g下在4℃下5分钟。 注:对于除本文所提出的四天协议更长的培养期间，细胞应大致使用相同的协议转染的每3天。第2步0.4应当但是修改，因为用不同的小RNA处理的细胞不应该被合并。所有的被测试的条件必须收集，计数，并单独转染。 </li><li>小心吸出上清液，然后重悬细胞中至少1毫升的PBS洗涤。计数活Th17细胞（ 例如 ，使用台盼蓝排除来评估活力血球），然后将细胞转移到一个离心管中。 </li><li>离心在500xg 5分钟在室温下使细胞沉淀。 </li><li>小心吸出上清液，然后使用从转染系统10μL试剂盒所提供的重悬缓冲液以每毫升2.5-4×10 7个细胞的密度悬浮细胞。保持细胞在室温下。 注:作为指导原则，尽量只准备尽可能多的细胞可以在30分钟内被转染。多批次可以比拟的。 </li><li>添加细胞的9μL到的小RNA 1μL在每个的MicroCentrifuge管。移液管一次混合用于转染细胞和小RNA然后加载到移液管提供电极尖端。 注意:通常情况下，添加细胞悬浮液的9.5μL，以确保有足够的混合物，以防止在转染前吸管电极末端产生气泡。 </li><li>用3mL室温电解缓冲液E.地点的吸液管站内的反应杯填充提供反应杯，然后将吸管到试管内的位置。 </li><li>立即电穿孔使用下列参数细胞和小RNA的各10μL混合物:脉冲电压1,500-1,550伏，脉冲宽度为10ms，和3个脉冲总转染装置上。 </li><li>电穿孔完成后，直接在细胞混合物添加到1X的Th17偏振媒体在一个培养板的孔中制备的500μL。代替板在5％CO 2，37℃的培养箱中两天。 注意:通过移液洗净移液管尖电极起来，并在PBS中向下每次转染之间。 </li></ol> 3.收获人Th17细胞<ol><li>重悬在培养基中的细胞微量，轻轻吹打，但要确保所有的细胞是从孔的底部分离。准备功能和/或基因表达分析的细胞。 注意:常规地，足够的细胞被转染的Th17细胞的单个孔中，得到用于表面标记物和细胞内细胞因子和转录因子染色的流式细胞术分析或用于制备RNA的用于基因表达分析。 </li></ol>

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The authors have nothing to disclose.

该协议提供用于递送的小RNA的成人Th17细胞的改进的方法。虽然人Th17细胞在此使用，电穿孔的这种方法与小RNA可以与其他主人T辅助细胞亚组，如TH1，TH2，以及调节性T细胞可以使用。它不是幼稚的CD4 + T细胞的运作良好，因此细胞必须培养的转染之前被激活。对于此协议中，我们首先优化了体外培养体系为更好的IL-17A的生产。最大的因素是基础的媒体。我们发现，无血清培养基优于传...

CD4 + T细胞是适应性免疫应答的关键协调器。幼稚CD4 + T细胞是能够发育成几个不同的效应T细胞（ 例如 TH1，TH2，Th17细胞， 等 ），每个具有自己的一套特性的细胞因子和转录因子，取决于局部微环境1。血统决定T细胞使对于两个保护性免疫和容忍，自我批评。 Th17细胞是公知的，以打击胞外细菌和真菌的T细胞的一个子集，但它们的不当响应也牵涉在多种自身免疫疾病和炎性疾病的发病机理，例如多发性硬化和牛皮癣2,3。人Th17细胞可以从幼稚的CD4 + T细胞在体外通过提供一个适当的偏振环境4来生成。 VARIO<html>我们的细胞因子的组合IL-1β，IL-23，TGFβ，和IL-6已被用于人Th17细胞的发展。人Th17细胞表达CCR6，即通常用于识别该细胞群，并通过它们的主要转录因子，RORγt（由RORC编码）5,6的表达被定义趋化因子受体。 Th17细胞具有表达多种细胞因子的能力，但IL-17A是由这些细胞产生的细胞谱系限定的效应细胞因子。我们检查了所有三个Th17细胞相关标记（CCR6，RORγt，IL-17A）的表达来评估我们的人Th17 体外分化检测的鲁棒性。此外，我们培养非偏振的条件下，在没有细胞因子或阻断抗体添加到培养基中作为阴性对照使用，因为这些Th17细胞标志物的表达应该是非常低的或不存在下的人CD4 + T细胞。 ve_content"&gt;学习正常的人T细胞的发育和生物学的一种方法是其发展过程中操纵基因表达。短干扰RNA（siRNA）是靶向蛋白质编码的mRNA，并且可以被利用来降低特定基因表达的合成的小RNA分子。微RNA（miRNA）是公知的调节基因表达的转录后的内源性非编码小RNA。的miRNA已显示出在小鼠和人的T细胞生物学中起重要作用，包括在Th17细胞7，8，9，它关键的是要具有在人T细胞操纵的小RNA的活性，研究其对基因表达，并最终对人T细胞生物学效应的可靠的方法。在此，我们描述了我们用于引入开发了一种易于使用的，一致的和可靠的协议小合成的RNA和锁定核酸（LNA，化学修饰的核酸具有增加的稳定性）成免疫细胞，并且具体到人Th17细胞。 有引入的小RNA到哺乳动物细胞中，其通常落入化学，生物或物理类别10的若干替代方法。常用的化学方法，包括基于脂质的转染和磷酸钙转染，依靠创建被细胞更有效地吸收化学-DNA复合物。一般而言，化学方法是不适合的原代T细胞的转染效率高。最常见的生物的方法是使用病毒载体（ 例如逆转录病毒或慢病毒），其直接插入外来RNA引入宿主作为其天然复制周期的一部分。病毒转导通常需要更长的时间来完成，特别是当一个在时间因素的原病毒的质粒分子克隆。此外，病毒转导载体可以是可能危害人体研究。电穿孔是一种物理米通过使细胞对高的电压脉冲，从而允许核酸以瞬时进入细胞在那里他们可以在他们的目标作用诱导膜通透性ethod。传统的电文书不能有效转染主要淋巴细胞。然而，优化的下一代电穿孔已经被证明是能够以很高的效率转染的T细胞，特别是当待转染的材料为小RNA。术语下一代是松散用来区分传统的电穿孔机两个新的平台（ 例如 ，霓虹灯，Amaxa公司）。此外，此方法是一种用于具有高达在单一实验中约120的小RNA，其通常使用验证合成试剂中等通量筛选容易扩展。重要的是，转染的成功可以在T细胞活化后只需16小时来实现。这种方法的缺点，但是，它不会导致基因组稳定incorporati上，因此是短暂的。因此，这是值得的额外的努力以创建可以被包装到病毒载体中并在T细胞在需要小RNA的长期表达的情况下成功地表达了稳定的表达构建体。 我们已经使用了下一代的转染（ 例如 ，氖），实现多样化合成的单链或双链RNA或LNA寡核苷酸的工具用于不同的目的11，12，13。有效的RNA干扰可以在原代小鼠和人T细胞使用双链短干扰RNA（siRNA）所诱导。这个协议描述了用于在人Th17细胞使用这种技术的优化条件。除了的siRNA，可商购的合成的miRNA模拟物和抑制剂可用于研究的miRNA增益和功能丧失。 miRNA模拟物是双链RNA分子非常相似的siRNA，但designe20d与内源成熟miRNA的序列。 miRNA的抑制剂是经化学修饰的结合天然miRNA和拮抗它们的功能的RNA和/或LNA基于单链寡核苷酸。我们发现，所有这些工具都可以在原代培养的T淋巴细胞有效地使用，包括但不限于人Th17细胞。

<table><tbody><tr><td>anti-human IL-17A PE</td><td>ebioscience</td><td>12-7179-42&amp;nbsp;</td><td>Clone: eBio64DEC17</td></tr><tr><td>anti-human IFNg FITC</td><td>ebioscience</td><td>11-7319-82</td><td>Clone: 4S.B3</td></tr><tr><td>anti-human CD4 eVolve605</td><td>ebioscience</td><td>83-0047-42</td><td>Clone: SK3</td></tr><tr><td>mouse anti-human CD196 (CCR6) BV421</td><td>BD biosciences</td><td>562515</td><td>Clone: 11A9</td></tr><tr><td>anti-human RORgt AF647</td><td>BD biosciences</td><td>563620</td><td>Clone: Q21-559</td></tr><tr><td>anti-human CD45 eFluor450</td><td>ebioscience</td><td>48-9459-42</td><td>Clone: 2D1</td></tr><tr><td>Foxp3/Transcription Factor Staining Buffer Set</td><td>ebioscience</td><td>00-5523-00</td><td>For intracellular transcription factor flow cytometry staining</td></tr><tr><td>Hu FcR Binding Inhibitor Purified</td><td>ebioscience</td><td>14-9161-71</td><td></td></tr><tr><td>ImmunoCult-XF T Cell Expansion Medium</td><td>Stemcell Technologies</td><td>10981</td><td>&quot;Serum-Free Base Media&quot;</td></tr><tr><td>MACS CD28 pure functional grade, human</td><td>Miltenyi Biotec</td><td>130-093-375</td><td>Clone: 15E8</td></tr><tr><td>anti-human CD3 Purified</td><td>UCSF monoclonal antibody core</td><td>N/A</td><td>Clone: OKT-3</td></tr><tr><td>LEAF purified anti-human IL-4</td><td>Biolegend</td><td>500815</td><td>Clone: MP4-25D2</td></tr><tr><td>anti-human IFNg, functional grade purified</td><td>ebioscience</td><td>16-7318-85</td><td>Clone: NIB42</td></tr><tr><td>Recombinant human IL-23</td><td>Peprotech&amp;nbsp;</td><td>200-23</td><td></td></tr><tr><td>Recombinant human IL-1&amp;beta;</td><td>Peprotech&amp;nbsp;</td><td>200-01B</td><td></td></tr><tr><td>Recombinant human TGF-&amp;beta;1</td><td>Peprotech&amp;nbsp;</td><td>100-21C</td><td></td></tr><tr><td>Recombinant human IL-6</td><td>Peprotech&amp;nbsp;</td><td>200-06</td><td></td></tr><tr><td>siGENOME Control Pool, Non-targeting #2</td><td>Dharmacon</td><td>D-001206-14-05</td><td></td></tr><tr><td>siGENOME SMARTpool Human RORC</td><td>Dharmacon</td><td>M-003442-00</td><td></td></tr><tr><td>siGENOME SMARTpool Human PTPRC</td><td>Dharmacon</td><td>M-008067-01</td><td></td></tr><tr><td>Dynabeads Untouched Human CD4 T Cells Kit</td><td>ThermoFisher Scientific</td><td>11346D</td><td>Human CD4&lt;sup&gt;+&lt;/sup&gt; T cell Isolation Kit</td></tr><tr><td>Neon Tranfection System</td><td>ThermoFisher Scientific</td><td>MPK5000</td><td>Next generation electroporation instrument</td></tr><tr><td>Neon Tranfection System 10 &amp;mu;L Kit</td><td>ThermoFisher Scientific</td><td>MPK1096</td><td></td></tr><tr><td>Resuspension Buffer T</td><td>ThermoFisher Scientific</td><td>Provided in kit (MPK1096)</td><td>&quot;Transfection Resuspension Buffer&quot;</td></tr><tr><td>Lymphoprep</td><td>Stemcell Technologies</td><td>#07801</td><td>Density Gradient Medium</td></tr><tr><td>costar 6-well tissue culture treated plates</td><td>Corning</td><td>3516</td><td>flat bottom plates</td></tr><tr><td>costar 48-well tissue culture treated plates</td><td>Corning</td><td>3548</td><td>flat bottom plates</td></tr><tr><td>BD Pharm Lyse lysing buffer, 10x</td><td>BD biosciences</td><td>555899</td><td>Must make 1x solution with distilled water prior to use</td></tr></tbody></table>

small rna transfection in primary human th17 cells by next generation electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

第一步，显影的成功电穿孔人Th17细胞的可靠系统是产生在体外分化的人Th17细胞培养物的鲁棒性。 Th17的偏振条件下培养的T细胞中表达的趋化因子受体CCR6和转录因子RORγt（ 图1A，左）。当T细胞非偏振（THN）的条件下（ 图1A，右图）培养没有表达这些标记物。 Th17的偏振条件下培养的T细胞也取决于再刺激产生的IL-17A（ 图1B，左侧...

Watch this Scientific Journal Video about Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation at JoVE.com

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

小RNA转染原代人Th17细胞通过下一代电穿孔

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

small-rna-transfection-primary-human-th17-cells-next-generation

Research

JoVE Journal

Immunology and Infection

7.8K 次观看.  University of California, San Francisco. Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

视频: Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

高效转染树突状细胞的无细胞的成熟优化的协议

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

科研

免疫与感染

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

高效率，站点特定的贴壁细胞转染siRNA的微电极阵列（MEA）

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

生物工程

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 ...

生物学

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

人THP-1巨噬细胞通过核转高效转染

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

利用 Cas9 Ribonucleoproteins 生成人 NK 细胞的研究

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 &#181;L of resuspension buffer and combined with 2 &#181;L of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

使用睡美人转座子系统对原代人色素上皮细胞进行基于电穿孔的遗传修饰

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately ...

医学

<meta charset="utf8"></head><body>使用流式细胞术从初始 CD4+ T 细胞中分化 Th17 细胞

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

<meta charset="utf8"></head><body>作者聚焦：使用细胞因子浓度调节实现 Th17 细胞的高纯度体外分化

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.

小鼠幼稚 CD4+ T 细胞体外分化为致病性 Th17 细胞

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

<meta charset="utf8"></head><body>向导 RNA 的逆转录病毒转导用于 Th17 分化中的基因编辑

Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay.
Conventional Th17 polarization methods typically involve obtaining na&#239;ve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate na&#239;ve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.

<meta charset="utf8"></head><body>逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17 differentiation.

逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells ...

小RNA转染原代人Th17细胞通过下一代电穿孔

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小鼠幼稚 CD4⁺ T 细胞体外分化为致病性 Th17 细胞

小鼠幼稚 CD4⁺ T 细胞体外分化为致病性 Th17 细胞

逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化