Name: optimal lentivirus production and cell culture conditions necessary to successfully transduce primary human bronchial epithelial cells
Uploaded: 2016-07-22T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells at JoVE.com

我们感谢迈阿密大学的生命器官联盟追回署提供的肺部。我们也感谢丽莎昆兹博士用于图2B和3-D，本Gerovac博士和丽莎·诺瓦克在图3A至C.用于实验的mCherry病毒所示的实验DNA准备，我们也感谢加布里埃尔Gaidosh从成像设备，眼科在迈阿密大学。 这项研究是由来自美国国立卫生研究院，在CF基金会和乘务员医学研究所的马蒂亚斯Salathe博士助学金资助。

1.第一阶段:HEK 293T文化<ol><li>制备HEK细胞培养基中添加10％（V / V）胎牛血清（FBS）和1％（体积/体积）青霉素/链霉素，以高糖的Dulbecco氏改良Eagle氏培养基（DMEM）（简称为HEK介质）。 </li><li>涂布一10厘米组织培养皿胶原I.混合30胶原微升我在2ml蒸馏水中。扩散混合到培养皿孵育在37℃下2小时和5％ 的 CO 2。 </li><li>取出混合物，并让在层流柜的菜干燥15分钟。 </li><li>板HEK细胞上以在10毫升的HEK媒体1×10 6个细胞的密度涂布培养皿孵育在37℃和5％的CO 2。 </li><li>当HEK细胞50-60％融合（ 图1a，电镀后2-3天），进行第2阶段的Visual汇合的评估就足够了。不需要定量。 注意:HEK细胞是在HEK培养基（见上文）。当他们到达汇合，T哎可以分割（ 例如 1/5）或冷冻下来以后使用。 </li></ol> 2.第二阶段:在HEK细胞生产和慢病毒（LVS）的浓度注:本协议应与机构和政府规章线以下的BSL-2在美国+（增强BSL-2）的条件下执行。 <ol><li>使用钙沉淀过程转染（脂质体和其它转染剂不如钙沉淀步骤，有效）。市售的转染试剂盒的用途，建议以保证再现性，虽然该过程修改如下。将所有试剂置于室温。这为0的一天。 </li><li>对于HEK293细胞每10 cm培养皿，搭配4.5微克包膜DNA PMD2-VSVG的，7.5微克包装DNA pMDLg / pRRE，3.75微克DNA PRSV-REV，慢病毒表达载体的10微克DNA，86微升的2M钙溶液和无菌水在一15ml离心管，以700微升的最终体积（在商业转染试剂盒中提供的后两个解）。 </li><li>逐滴添加2×HEPES-缓冲盐水的700微升（HBS，在转染试剂盒中提供），而涡旋（在层流柜）并在室温下温育30分钟（执行步骤4在等待）。 </li><li>在早期的孵化时间，定金5微升幻灯片上的搭配。检查液滴是10X的目标，在显微镜下，重点慢慢向上和向下。一种细的沉淀物应该是可见的，证实了磷酸钙沉淀法（ 图1b）。 </li><li>通过加入10ml的HEK细胞介质和移液器上下的混合停止30分钟后的沉淀。 </li><li>以HEK细胞的培养皿步骤2.1-2.5，并取出介质（0天）。小心缓慢地添加来自步骤2.5的沉淀溶液沿着盘的边缘。 </li><li>次日（第1天），除去并丢弃培养基含有沉淀并更换瓦特第i个10毫升HEK培养基。检查在显微镜下精细沉淀物:它应该是在细胞（ 图1C和D）之间的空白空间的菜可见。 </li><li>第2天，收集培养基用注射器，过滤器通过0.45μm针筒过滤器进含有3.8毫升40％聚乙二醇（PEG）溶液15毫升离心管中。倒置5倍以混合并储存在4℃下至少24小时（对于PEG沉淀形成，但超过5天不再），直到所有的病毒集合是完整的。 </li><li>天3和4。第4天，重复步骤2.8不HEK中等取代但10％的漂白粉消毒和丢弃的菜。 </li><li>离心机所有收集管（通常在第5天在一起的所有集合）于1650×g离心20分钟，在4℃以沉淀病毒。除去并弃去上清液，并在4℃下于1650 xg离心再次旋转5分钟以收集和去除的PEG上清的任何剩余。 </li><li> Resus挂起在200μl支气管上皮生长培养基（BEGM）的每个管的无两性霉素B 8沉淀。池他们在一起，并在200微升等分试样。在-80°C储存病毒。分装额外的10微升进行HIV-1加格（P24）抗原ELISA检测。 </li><li>使用商业ELISA试剂盒，根据制造商的协议来执行一个p24抗原测定。该试验给出了以pg / ml的病毒的p24的估计。 </li></ol> 3.第三阶段:培养初HBE细胞<ol><li>在10ml BEGM介质板HBE细胞（用100μl两性霉素B，如果通道0细胞用于消除可能的真菌污染）在我涂覆10厘米组织培养皿胶原一个1×10 6个细胞的密度。在37℃和5％的CO 2。 </li><li>次日，除去培养基并用10ml BEGM取代（用100μl两性霉素B为通道0细胞），共4天。返回到孵化器。 </li><li>经过4天AYS，只使用不BEGM两性霉素B更改媒体隔日1次。 </li><li>当细胞为80-90％汇合（ 图2a;〜铺板后7天），则继续转导（注意3.5用于制备转导之前）。再次，汇合的视觉评估是足够的。不需要定量。 </li><li>前转导，外衣用IV型胶原溶液的透气性支承插入物稀释1/10用无菌蒸馏水。加入200μl到每个插入，并让在层流柜干燥过夜。 </li></ol> 4.第四阶段:支气管上皮细胞转导<ol><li>除去介质，用磷酸盐缓冲盐水（EDTA / PBS）中加入3毫升0.1N％胰蛋白酶在1mM的乙二胺四乙酸和孵育在37℃5分钟和5％ 的 CO 2。检查显微镜（10X物镜）下，看看是否所有单元被分离。如果没有，返回到培养箱中另外5分钟。 </li><li>当所有的细胞分离，加入3毫升大豆胰蛋白酶抑制剂（SBTI 1毫克/毫升），混合并转移至15毫升离心管中。通过在460 xg离心纺丝在室温下5分钟沉淀细胞。 </li><li>除去上清液，重悬沉淀于3ml BEGM并具有计数继续。 </li><li>悬浮HBE细胞（现在通道1或P1）以2×10 5个细胞每112毫米透气性支承插入于400μlBEGM的（ 见表2）的比率。推断基于将要转导的透气性支承插入物的量这些数字。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 表面 </td><td> 表面积 </td><td> 细胞计数 </td><td> 媒体 </td><td> 体积（媒体） </td></tr><tr><td> 10cm皿</td><td> 52.7厘米2 </td><td> 1×10 6个 </td><td> BEGM </td><td> 10毫升</td> &lt;/ TR&gt; <tr><td> 12毫米过滤器</td><td> 1.13厘米2 </td><td> 2×10 5个 </td><td> BEGM </td><td> 400微升</td></tr><tr><td> 24毫米过滤器</td><td> 4.52厘米2 </td><td> 8×10 5个 </td><td> BEGM </td><td> 800微升</td></tr></tbody></table> 表2:每个细胞计数媒体推断。 <ol start="5"><li>使用的4感染复数（MOI）因子的多个，添加病毒的适当体积的细胞悬浮液。 </li><li>聚凝胺添加至2微克/毫升的最终浓度。 </li><li>免除400微升每透水支持插入混合（BEGM，HBE细胞，病毒和聚凝胺）进入顶部隔室，独自加入1ml BEGM到基底仓。在37℃和5％的CO 2总是板HBE细胞孵育过夜无病毒作为对照和测试嘌呤选择（如果适用）。 </li><li>第二天，对物OVE根尖和基底外侧培养基，用PBS洗，并替换为空气-液体界面（ALI）媒体8两个室。 </li><li>当细胞汇合，除去心尖流体（称为建立气 - 液界面）。继续隔日舱底部改变介质（ALI），直到他们达到完全成熟;通常为3至4周后。 注意:如果慢病毒载体含有一个选择基因，如嘌呤霉素，细胞可以通过根据选择浓度曲线加入嘌呤霉素进行选择。对于HBE细胞，1微克/ ml的浓度被确定为理想的转导的细胞的一致的选择。然而，这个浓度可能会有所不同其他细胞或条件，应由杀死曲线来确定。媒体已经更换后ALI媒体或更高版本（2-3天），允许选择基因充分表达嘌呤霉素的加入可以开始，早一天。一定要嘌呤霉素添加到一个插入使得h因为没有被转导。可以加入嘌呤霉素直至细胞完全分化。 </li></ol>

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The authors have nothing to disclose.

这里所概述的协议保证接近100％转导效率。但是，也有在此过程中是实现这一目标的重要步骤。例如，在转染过程中，析出物中的转导结构的存在（下降），或在培养皿后转导（ 图1b和d）该天是一个良好的转染都相关。没有沉淀物或附聚物的存在可能是由于在转染试剂中的一个的遗漏，pH值的问题，或坏质粒制备。如果沉淀缺失，有很高的可能性，没有将可见的第二天。...

复制缺陷型，假慢病毒的发展（LV）也为研究人员提供一个安全和有效的方法， 在体外 1提供转基因。由于它们的长期表达，这些LV也可以用于治疗剂的体内 2,3的输送。生产的LV需要人胚胎肾（HEK）细胞的几个质粒共转染:转移载体，包装的gag / POL，包装转速 ，和包络水泡性口炎病毒糖蛋白（VSV-G）的质粒。第三代包装系统被认为比第二代系统更安全，因为转速基因被编码在一个单独的包装质粒，从而降低重组的可能性，以产生具有复制能力的慢病毒。虽然第二代包装系统收率五倍高的总转导单元，原病毒基因组的分割以创建第三代系统已降低转导的水平仅最低限度3,4。 而细胞系可更容易和有效地转导，研究人员已经将重点转向主细胞，因为这些是更具有代表性的体内组织中5,6。然而，原代细胞是难治转导。而完全分化呼吸道上皮细胞的转导已经报道，整体效率还没有超过15％的7。 这里描述的是一个协议，允许生产高滴度的LV的。一个一步一步的方法概述实现近100％的转导效率分为原发性支气管上皮细胞。更具体地，该协议描述器乐成功3的最佳培养条件。简言之，HEK 293T细胞使用的是带有增强型绿色荧光的EF-1启动子驱动表达慢病毒载体转染PCDH蛋白（EGFP）或mCherry，红色荧光蛋白，和一个第三代包装系统。释放到介质的LV被收集24至72小时后。病毒颗粒集中用聚乙二醇（PEG），病毒浓度的方便和简单的方法，采用了p24抗原的酶联免疫吸附测定（ELISA）试剂盒滴度估计之前。随后，未分化的初级HBE细胞被转导在增殖培养基（支气管上皮生长培养基或BEGM）8，而安装（被胰蛋白酶处理后），在感染复数的4（MOI）因子，加入聚凝胺和温育16小时（过夜）。然后允许细胞分化。由于病毒的转基因被表达长期（使用适当的启动子，见讨论），表达将在整个分化维持成假呼吸道上皮或可以（使用诱导型启动子）的分化后诱导。

<table><tbody><tr><td>HEK293T/17 cells</td><td>ATCC</td><td>CRL-11268</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td>Sigma</td><td>12306C</td><td>use at 10%</td></tr><tr><td>DMEM</td><td>Thermo Scientific</td><td>11995-040</td><td></td></tr><tr><td>Penicillin/Streptomycin (100x)</td><td>Thermo Scientific</td><td>15140-122</td><td>use at 1x</td></tr><tr><td>Collagen I</td><td>BD Biosciences</td><td>354231</td><td>dilute 1:75 in distilled water</td></tr><tr><td>Calphos</td><td>Clontech</td><td>631312</td><td>Transfection kit containing 2 M Calcium solution, 2x HEPES-Buffered solution (HBS) and sterile H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Polythylene glycol 8000</td><td>VWR scientific</td><td>101108-210</td><td>stock of 40%</td></tr><tr><td>Amphotericin B</td><td>Sigma</td><td>A9528</td><td>use at 1x</td></tr><tr><td>Trypsin</td><td>Sigma</td><td>T4799</td><td></td></tr><tr><td>Soybean trypsin inhibitor</td><td>Sigma</td><td>T9128</td><td></td></tr><tr><td>Transwell 12 mm</td><td>Corning</td><td>3460</td><td></td></tr><tr><td>Collagen IV</td><td>Sigma</td><td>C7521</td><td>dilute 1:10 in distilled water</td></tr><tr><td>Hexadimethrine bromide</td><td>Sigma</td><td>H9268</td><td>stock of 2 mg/ml</td></tr><tr><td>Puromycin (10.000x)</td><td>Thermo Scientific</td><td>A11138-03</td><td>use at 1x</td></tr><tr><td>Alliance HIV-1 p24 ELISA</td><td>PerkinElmer</td><td>NEK050001KT</td><td></td></tr><tr><td>F12</td><td>Thermo Scientific</td><td>11765-054</td><td></td></tr><tr><td>pCDH-EF1-MCS-IRES-Puro</td><td>System Biosciences</td><td>CD532A-2</td><td>lentiviral expression vector</td></tr><tr><td>pMD2-VSVG</td><td>Addgene</td><td>12259</td><td>packaging DNA</td></tr><tr><td>pMDLg/pRRE</td><td>Addgene</td><td>12251</td><td>packaging DNA</td></tr><tr><td>pRSV-rev</td><td>Addgene</td><td>12253</td><td>packaging DNA</td></tr></tbody></table>

optimal lentivirus production and cell culture conditions necessary to successfully transduce primary human bronchial epithelial cells

在使用空气-液体界面条件下提供了一个有用的模型来研究气道细胞分化和功能的处理原代人支气管上皮（HBE）细胞的体外培养。在过去的几年中，为转基因递送中使用的慢病毒载体的成为普遍的做法。虽然有完全分化的呼吸道上皮细胞与某些非HIV伪类型的慢病毒转导的报道，整体转导效率通常低于15％左右。这里介绍的协议提供了可靠和有效的方法，以产生慢病毒和转导原代人支气管上皮细胞。用未分化的支气管上皮细胞，支气管上皮生长培养基转导，而细胞附着，具有4感染因子的多个提供接近100％的效率。本协议描述的，一步一步的，高滴度的慢病毒载体及日的准备和浓度Ë传导过程。它讨论了确定的最佳培养条件，以实现初级人支气管上皮细胞的高效转导的实验。

图1描绘了在转染过程评估细胞和溶液的不同的关键步骤。 图1A示出的HEK293T细胞的转染病毒的产生之前最佳汇合。重要的是，将细胞在整个培养皿均匀分布。 图1b中揭示了在使用明视野光学系统和一个10X物镜的转染混合物一滴沉淀。越的析出物像砂粒，而不是附聚物，更有效的转染将。在这张图片中，沉淀物是有点粗糙。该沉淀物是?...

Watch this Scientific Journal Video about Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells at JoVE.com

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

优化慢病毒生产和细胞培养必要条件，成功转导初级人支气管上皮细胞

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

optimal-lentivirus-production-cell-culture-conditions-necessary-to

Research

JoVE Journal

Biology

19.2K 次观看.  University of Miami, Miller School of Medicine. Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

视频: Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

包装艾滋病毒或FIV的基于Lentivector表达结构与VSV-G假型病毒颗粒转导

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

科研

免疫与感染

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

协议小鼠胚胎成纤维细胞直接转化为滋养层干细胞

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

发育生物学

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

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

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

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 ...

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation.

小鼠髓质前体 32-脑脊液-R 细胞的增殖和分化

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

从外植体生长的首要人权支气管上皮细胞

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

生物学

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

增强生物安全与聚合物复合物Ecotropic慢病毒的人体细胞的转导

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

从高致病性H5N1病毒和禽流感病毒中生产含有包络糖蛋白的高蒂特感染性流感伪型颗粒

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in na&#239;ve human populations.

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

用于研究呼吸道病毒感染期间先天免疫细胞活化的非接触式共培养模型

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.