笔者想承认迈克尔·周，戴冰冰，梁萧此稿件提供数据。我们也承认约翰·格雷博士在这项研究中所使用的试剂厚礼。 PB是由国立癌症中心博士后奖学金支持。这项研究是由来自美国国立卫生研究院（R01AI68978 P01CA132681 RCA170820A），比尔和梅林达·盖茨基金会，平移加速转化医学联合中心和加州艾滋病毒/艾滋病的资助赠款的资助拨款研究计划。

DC-LV稳定生产细胞构造基于对GPR包装细胞系11包含必要的慢病毒组件gagpol，转和TET关闭系统。首先，逆转录病毒转导是用来产生一个GPRS编码一个四面体依赖SVGmu的糖蛋白的包装细胞系。然后，连环体数组转染用于转染的GPRS慢病毒载体的转基因如GFP细胞系。指定为LV-mGFP的这种稳定的生产细胞系，可在体外和体内进行测试，它能够产生一个DC-LV疫苗的GFP。 1。生成一个春节依赖SVGmu的细胞系质粒PRX-SVGmu是一个构建体，其中被克隆的DC-特异性糖蛋白SVGmu的的下游启动子的逆转录病毒质粒pRetroX四环素销1（ 图1C），tTA的先进。文化293T细胞在D10（Dulbecco改良的Eagle培养基与10％胎牛血清，2mM L-谷氨酰胺）。文化GPR与多西环素（1毫微克/毫升）和嘌呤霉素（2微克/毫升）在D10的包装细胞系。所有细胞培养在5％CO 2，饱和湿度37℃培养箱。 <ol><li>瞬时转染前16-18小时，板2×10 6 293T细胞在4ml的D10在6厘米的组织培养皿中，例如，细胞在转染时的接近90％汇合。 </li><li> 逆转录病毒质粒转染 :混合100微升1.25M的CaCl 2的溶液，无菌Milli-Q水和下列质粒:5微克PRX-SVGmu的，2.5微克pGag聚合物，2.5微克pVSV-G。最终体积为500微升。 5毫升聚苯乙烯圆底管，加入500μl2X HBS（50毫米HEPES，氯化钾10毫米，12毫米葡萄糖，氯化钠280毫米，1.5毫米的Na 2 HPO 4 * 7H 2 O，pH值7.05）。添加质粒进入2X HBS缓冲区的混合物滴鼓泡缓冲区的同时大力巴斯德玻璃吸管。添加英里后夹具，继续冒泡另一个30秒。然后，将整个混合物添加到293T细胞在6厘米培养皿中，在37℃下</li><li> 4小时后，转染，仔细更换介质在培养皿中，用4毫升预热D10。 </li><li> 自旋感染 :在转染后48小时，收获SVGmu编码，在上清液中的逆转录病毒颗粒的上清液通过0.45μm的过滤器通过。 GPR包装板在24孔板中的细胞在2×10 4个细胞/孔。添加过滤上清液GPR包装细胞中的菜（2毫升/孔），离心细胞90分钟，以1050×g，25℃下更改介质到新鲜的D10与强力霉素（1纳克/毫升）后旋感染（2毫升/）。 </li><li>转染后72小时，扩大文化转D10用强力霉素（1纳克/毫升）和嘌呤（2毫微克/毫升）包装细胞。 </li><li>要确认SVGmu，培养细胞的表达没有强力霉素48小时。测量表面前的SVGmu PRESSION流式细胞仪，使用抗辛德毕斯血清。这些细胞被指定为GPRS包装细胞系。 </li></ol> 2。构建DC LV生产者细胞连环阵转慢病毒转移质粒TL20-GFP的是一个自失活慢病毒转移载体质粒基于pCL20c MSCV-GFP一个霉素调节的病毒RNA基因组的表达系统和巨细胞病毒（CMV）的增强子与7 四面体运营商（ 图1C）11更换，12,15。质粒PGK-BLE是一种抗博莱霉素（BLE）盒式磁带驱动由弱PGK启动子2。 <ol><li>月刊20微克质粒TL20 SFII GFP用限制性内切酶在50℃下精华20微克质粒PGK-BLE PflMI位在37°C。 </li><li>通过琼脂糖凝胶电泳纯化DNA片段（PGK表盒1011 bp和矢量TL20-GFP为6861碱基对）。 </li><li>结扎TL20-GFP载体和PGK-表盒（NEB的T4 DNA连接酶）的摩尔比为25:1。在室温下孵育过夜。 </li><li>隔夜后结扎，纯化DNA连接混合物（Qiagen公司的DNeasy血液和组织工具包）。确保纯化的DNA的量为约5微克。 </li><li>转染前16-18小时，板GPRS包装细胞在6厘米的组织培养皿中，使得将约90％汇合的转染时。 </li><li>使用在步骤1.2中所述的纯化的concatemeric DNA转染5微克到GPRS包装细胞在6厘米的组织培养皿中的磷酸钙转染方法。 4小时后，转染，小心取出介质，并更换有4毫升预热D10采用强力霉素（1纳克/毫升）。 </li><li>转染后48小时，更改到D10中的介质与多西环素（1毫微克/毫升）和嘌呤霉素（2微克/毫升）。吉欧霉素（50微克/毫升）在培养基中加入转染克隆的选择。培养细胞约2周，直到细胞科洛尼ES可以看出底部的菜肴。 </li><li>标记的细胞集落，在底部的菜肴。径24孔组织培养皿中，数量的孔，并加至各孔中的2毫升D10含有Zeocin的（50微克/毫升），多西环素（1毫微克/毫升），嘌呤霉素（2微克/毫升）。吸媒介转染细胞，然后添加一个胰蛋白酶下降到不到一分钟，每个殖民地。然后，添加一个或多个下降的D10相同的菌落。拿起菌落逐个用吸管，并将它们传送到单独的井24孔细胞培养板。 </li><li>文化和扩大D10含有Zeocin的（50微克/毫升），多西环素（1毫微克/毫升）和嘌呤霉素（2微克/毫升）中的所有细胞克隆的病毒产生能力的评价。 </li></ol> 3。评估病毒生产的每一个细胞克隆<ol><li>胰蛋白酶消化的生产者细胞和板周围4×10 6 6-D10厘米的组织培养皿中的细胞在没有强力霉素等汇合超过90％。每天用新鲜预热D10更换介质和收集介质滴度检测72小时，取出后DOX。 </li><li>收获的病毒上清用0.45微米的过滤器通过过滤介质。 </li><li>板靶细胞表达DC-SIGN受体（ 如 293T.hDC-SIGN或小鼠骨髓来源的树突状细胞的16）和293T细胞作为阴性对照，在96孔培养皿中，1×10 4个细胞/孔。 2倍系列稀释的病毒上清添加到各孔中（100μl/孔）。 ，6-8的稀释度一般足以达到在其中转导的GFP阳性细胞是在添加的病毒量成线性关系的范围内。 </li><li>离心细胞，更换介质，在步骤1.4中所述。 DC培养上清应培养在RPMI培养基中含10％FBS和GM-CSF（1:20 J558L条件培养基）。 </li><li>测量GFP表达的转染细胞，通过流式细胞仪检测转导后4-6天。慢病毒的VECTO的ŗ滴度计算向量中的稀释范围内时，GFP阳性细胞的百分比和矢量量的线性关系。具有最高的病毒生产用于后续的应用中选择的细胞克隆。 </li></ol> 4。生产和集中慢病毒载体<ol><li>文化生产者细胞系（LV-mGFP的），在15厘米的组织培养皿。 </li><li>胰蛋白酶消化的的生产者细胞和板细胞在15厘米的组织培养皿中，在大于90％汇合在多西环素的新鲜D10无。更改介质每日新鲜的，预先加热D10。 </li><li>峰值病毒生产（由用户确定）的时间，收获病毒上清，用0.45微米的过滤器过滤。装入过滤上清液成厚壁32.5毫升超速离心管，封口的封口膜和离心机以50,000 xg，4°C，90分钟。彻底将沉淀重悬于50μl或适当体积的PBS或HBSS中，根据不同的应用。&lt;/ LI&gt; </ol> 5。免疫小鼠体内，并分析抗原特异性免疫反应<ol><li>第4节所述产生高滴度的病毒。 </li><li>通过载体悬浮（25微升每脚垫）脚垫路由注入BALB / c小鼠（6-8周龄，女）。 </li><li>免疫后2周，隔离健脾和收获脾。培养脾细胞用GFP的表位肽和分析GFP特异的CD8 + T细胞，使用细胞内细胞因子染色的存在下，如前面所述17（ 图3B）。 </li></ol>

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<p class="jove_content"&gt;作者宣称，他们有没有竞争经济利益。</p

在这里，我们已经提出了一个方法生产大量使用慢病毒载体293T细胞稳定转关监管制度下TET所有慢病毒组件。至目前为止，大多数协议产生慢病毒载体依赖于标准的磷酸钙瞬时转染（见18）。这种方法已成功地在一个临床范围，但它可能遭受的一些限制，不太可能是存在于扩大生产的稳定细胞株。例如，与大量的LV质粒的共转染理论上可以增加复制能力的慢病毒19的发展机会。此外?...

已经开发了许多载体系统的基因传递。基于慢病毒载体已跻身研究最常用的病毒系统。这些载体是有利的，因为他们可以高效转染分裂和非分裂细胞1，由于整合到宿主基因组实现长期表达，表现出低的天然抗载体在大多数人群的免疫力，并具有低潜力从插入突变3,4的遗传毒性。 慢病毒的生产安全问题一直着色。慢病毒载体一般是来自于HIV-1，艾滋病的病原体。单个元件的慢病毒（转让，外壳和包装质粒）的瞬时转染是一种常见的和灵活的方法，在实验室设置中提供的遗传物质。然而，扩大规模的临床应用瞬时转染是繁琐和马的Ÿ导致复制能力的慢病毒5,6的发展。要克服这些障碍，稳定的包装和生产细胞系已开发6-11。这些线路之一，探地雷达的包装生产线11，受四环素有吸引力的优势。在本文中，我们将演示如何适应这种系统产生自我失活的慢病毒载体，是专门面向树突状细胞（DC-LVS）12。 树突状细胞（DC）是最强大的抗原呈递细胞的免疫系统。他们已经在癌症疫苗开发的主题极大的兴趣，因为它们直接启动，程序和规范肿瘤特异性免疫反应13。装有包括区议会有可能引出更强的抗肿瘤免疫反应比肽或DNA疫苗的接种方案。最近，我们已经开发出一种慢病毒载体，specificallÝ针对树突状细胞，通过修改辛德毕斯病毒糖蛋白，SVGmu 14。这些载体是独特的，他们表现出高特异性树突状细胞，并产生更强的免疫反应比特异性，VSVG假型载体。 在这里，我们报告的方法产生大量的这些直流针对性的慢病毒载体。我们表明，这些DC-LVS可以感染区议会和产生强烈的CD8 + T细胞的免疫反应。所有程序涉及动物的人道待遇，执行下USC Intitutional动物护理和使用委员会批准。为了执行在体内和临床实验，这是至关重要的建立的细胞系，可以制作高滴度的病毒。执行转导和转染与所述的相同的步骤，将产生这样的克隆的机会最大化。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Mediatech Inc.</td>
			<td>10-013-CV</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glutamine</td>
			<td>Mediatech Inc.</td>
			<td>25-005-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Doxycycline</td>
			<td>Clontech Laboratories, Inc.</td>
			<td>631311</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Zeocin</td>
			<td>Invitrogen</td>
			<td>R250-01</td>
			<td>Toxic</td>
		 </tr>
		 <tr>
			<td>Puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td></td>
		 </tr>
		 <tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DNeasy Blood &amp; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a tetracycline-regulated cell line produces high-titer lentiviral vectors that specifically target dendritic cells

慢病毒载体（逻辑卷）是许多类型的细胞提供遗传材料的有力手段。由于相关的安全问题与这些HIV-1的衍生载体，产生大量的LVS是具有挑战性的。在本文中，我们报道了一种用于生产高滴度自我失活LVS的。逆转录病毒转导的tet过稳定线GPR生产细胞产生的细胞系，GPRS，它可以表示所有的病毒组分，包括树突状细胞特异性糖蛋白，SVGmu。然后，我们使用concatemeric的LV的转移质粒编码DNA转染，转染报道基因GFP的组合用一个可选择的标记。得到的克隆可以制作LV上面滴度大于10倍的我们实现与瞬时转染的。另外，这些病毒高效转染树突状细胞在体外和产生强烈的T细胞免疫反应向本报记者抗原。这种方法可能是一个不错的选择FOŗ强LV为基础的疫苗，癌症或传染病的临床研究。

在该方法中所述的稳定的细胞系，可以专门针对树突状细胞的慢病毒载体，大批量地生产。 图1B中所示，分离单个克隆，得到的稳定细胞株的不同的质量12。在测试的26个克隆中，有8产生的慢病毒颗粒滴度大于10 6转导单位每毫升（TU /毫升），这是一个典型的为SVG的假型化慢病毒载体瞬时转染产生的基准。在同一时间，几个克隆产生小于10 4 TU /毫升，隔离是很?...

Watch this Scientific Journal Video about A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells at JoVE.com

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

这里，我们用逆转录病毒转导和concatemeric的转染建立的细胞系中能表达的慢病毒载体（LV）的组件在没有四环素。此LV编码绿色荧光蛋白是一种糖蛋白，SVGmu，这是具体的树突状细胞上的受体与假。

四环素调节细胞株产生高滴度的慢病毒载体，专门针对树突状细胞

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

a-tetracycline-regulated-cell-line-produces-high-titer-lentiviral

Research

JoVE Journal

Immunology and Infection

14.5K 次观看.  University of Southern California, Los Angeles. 这里，我们用逆转录病毒转导和concatemeric的转染建立的细胞系中能表达的慢病毒载体（LV）的组件在没有四环素。此LV编码绿色荧光蛋白是一种糖蛋白，SVGmu，这是具体的树突状细胞上的受体与假。

视频: A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

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 ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

遗传学

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

神经科学

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

（TET-ON）与四环素诱导shRNA或cDNA表达生成稳定的人类细胞系

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

生物学

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor (CAR)-T cells. These modified cells have shown remarkable clinical responses in patients with hematologic malignancies. However, the high cost of producing these therapies and conducting extensive quality control assessments has limited their accessibility to a broader range of patients. To address this issue, many academic institutions are exploring the feasibility of in-house manufacturing of genetically modified cells, while adhering to guidelines set by national and international regulatory agencies.
Manufacturing genetically modified T cell products on a large scale presents several challenges, particularly in terms of the institution's production capabilities and the need to meet infusion quantity requirements. One major challenge involves producing large-scale viral vectors under Good Manufacturing Practice (GMP) guidelines, which is often outsourced to external companies. Additionally, simplifying the T cell transduction process can help minimize variability between production batches, reduce costs, and facilitate personnel training. In this study, we outline a streamlined process for lentiviral transduction of primary human T cells with a fluorescent marker as the gene of interest. The entire process adheres to GMP-compliant standards and is implemented within our academic institution.

This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.

符合GMP的基因修饰T细胞生成程序

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor ...

生物工程

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro

Dendritic cells (DCs) are important antigen-presenting cells that connect innate and adaptive immune responses. DCs are heterogeneous and can be divided into conventional DCs (cDCs) and plasmacytoid DCs (pDCs). cDCs specializes in presenting antigens to and activate na&#239;ve T cells. On the other hand, pDCs can produce large quantities of type I interferons (IFN-I) during viral infection. The specification of DCs occurs at an early stage of DC progenitors in the bone marrow (BM) and is defined by a network of transcription factors (TFs). For example, cDCs highly express ID2, while pDCs highly express E2-2. Since more and more subsets of DCs are being identified, there is a growing interest in understanding specific TFs controlling DC development. Here, we establish a method to screen TFs critical for DCs differentiation in vitro by delivering lentivirus carrying short hairpin RNA (shRNA) into an immortalized hematopoietic stem and progenitor cell (iHSPCs) line. After the selection and in vitro differentiation, cDC and pDC potential of the stable knockdown cell lines are analyzed by flow cytometry. This approach provides a platform to identify genes potentially governing DC fates from progenitors in vitro.

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro

Here we provide a protocol for screening potential transcription factors involved in the development of dendritic cell (DC) using lentiviral transduction of shRNA to obtain stable knockdown cell lines for in vitro DC differentiation.

短发夹RNA介导的基因敲低在造血干和祖细胞系体外发育的树突状细胞发育研究

Dendritic cells (DCs) are important antigen-presenting cells that connect innate and adaptive immune responses. DCs are heterogeneous and can be divided ...

<meta charset="utf8"></head><body>用于体内基因调节的慢病毒载体制备

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue&#45;Resident Macrophages In Vivo in Mice

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

<meta charset="utf8"></head><body>作者聚焦:通过慢病毒载体介导的基因操作探索巨噬细胞免疫代谢

We demonstrate a step-by-step protocol for the investigation of gene function in peritoneal tissue-resident macrophages in vivo, using lentiviral vectors.

小鼠 体内 腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring ...

四环素调节细胞株产生高滴度的慢病毒载体，专门针对树突状细胞

摘要

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此视频中的章节

T细胞受体在小鼠T细胞的逆转录病毒转导

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

高效转染树突状细胞的无细胞的成熟优化的协议

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

从中枢神经系统转导细胞的慢病毒载体生产

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

（TET-ON）与四环素诱导shRNA或cDNA表达生成稳定的人类细胞系

符合GMP的基因修饰T细胞生成程序

短发夹RNA介导的基因敲低在造血干和祖细胞系体外发育的树突状细胞发育研究

小鼠体内腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

四环素调节细胞株产生高滴度的慢病毒载体，专门针对树突状细胞

摘要

探索更多视频

此视频中的章节

T细胞受体在小鼠T细胞的逆转录病毒转导

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

高效转染树突状细胞的无细胞的成熟优化的协议

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

从中枢神经系统转导细胞的慢病毒载体生产

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

（TET-ON）与四环素诱导shRNA或cDNA表达生成稳定的人类细胞系

符合GMP的基因修饰T细胞生成程序

短发夹RNA介导的基因敲低在造血干和祖细胞系体外发育的树突状细胞发育研究

小鼠 体内 腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

小鼠体内腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备