我们感谢我们实验室的所有成员的有益讨论。我们感谢的乔安娜Lisztwan和克里斯蒂娜Gewinner批判性阅读的手稿。本工作受的BBSRC授予BB/I021248/1和威康信托基金会资助090090/Z/09/ZAH是一个威康信托基金会的研究生涯发展的伦敦大学学院癌症研究所的研究员。

1。克隆pcDNA6_TetR_IRES_blast <ol><li>正如图3中所示，执行pcDNA6/TR质粒（V1025-20，Invitrogen公司）用限制性酶XbaⅠ和Nco I位删除的四环素基因和启动子的杀稻瘟素的电阻（BlastR）基因的部分的摘要。隔离4.6 kb的载体片段。 </li><li>要删除从四环素基因的多聚腺苷酸化序列，通过PCR引入XhoⅠ位点后，立即停止密码子，四环素，同时节省的Xba I位点的cDNA的5&#39;末端的cDNA。使用限制性内切酶XbaⅠ和XhoⅠ位生成pMigR1_TetR质粒亚克隆所得片段插入pMigR1 17，。 </li><li>消化pMigR1_TetR载体用限制性酶XbaⅠ和Nco I位，释放TetR_IRES片段。隔离1.25 kb的插入片段。 </li><li>结扎：4.6 KB pcDNA6/TR和1.25 kb的TetR_IRES片段。风帆连接产物ansform主管大肠杆菌，并使用标准的分子生物学技术确定所需的pcDNA6_TetR_IRES_blast结构。 </li></ol> 2。代稳定表达的细胞系四环素<ol><li>在第1天，种子1×10 6个细胞每10厘米的组织培养板中，使用标准的生长培养基（ 例如，用10％FCS的DMEM培养液）。种子两块板，一个用于转染pcDNA6_TetR_IRES_blast，和其他的杀稻瘟素的选择作为阴性对照。确保使用最低的通道可能和推荐的生长培养基中。 </li><li>在第2天，转染2微克pcDNA6_TetR_IRES_blast使用Fugene 6（E2691，Promega）中，按照生产商的说明的一个板。不要转染第二板。 </li><li>第3天，标准生长介质中含有5微克/毫升杀稻瘟到这两个板块。请注意，对于一个给定的细胞系的最佳选择的浓度可能会有所不同，可能Ň电火工品必须预先确定。 </li><li>在第4天，细胞分裂，在1/5，1/25，1/50和1/100使用10-cm的板和标准的生长培养基中含有5毫克/毫升的杀稻瘟素。确保正确标注板未转染或转染的细胞。准备两个板块每股稀释步骤。 </li><li>每天检查细胞，确保所有细胞的转染板的第一个星期内相继去世。更改选择媒体每隔2-3天。 </li><li> 1-2周后，杀稻瘟素抗性集落开始出现。选择板，含有5〜50的菌落，以确保单个菌落可以拾取。 </li><li>当菌落已经达到了一个直径大约5毫米（或更大），隔离至少12个独立的克隆，使用克隆缸挑选单个菌落。每个克隆转移到一个单独的24孔组织培养板的孔。当融合时，从每个孔中的细胞分裂成一个单一的孔的6孔组织培养板中。 </li><li>继续文化CL在选择媒体的。当融合时，从每个孔中的分裂细胞分为两个单独的6孔组织培养板的孔中。 </li><li>对于每个要测试的克隆，冻结一个孔的融合细胞和收获细胞，通过免疫印迹法的其他以及随后的测试。 </li><li>通过Western印迹法使用小鼠单克隆抗TET02抗体（MoBiTec联系）检测四环素。请记住，包括作为阴性对照的亲代细胞系的裂解物。 </li><li>四环素表达最高和扩大选择3个克隆，每一个克隆培养。尽快冻结股票的每一个有前途的克隆。最后，通过比较亲本细胞系与四环素表达克隆研究的分子和细胞生物学参数。选择“最好”的克隆与四环素表达最高不显示任何改变的分子和细胞生物学参数。 </li></ol> 3。试点测试pTER和PT-REX DEST30载体的表达ING shRNA或的cDNA， <ol><li>生成pTER的shRNA插入质粒的说明12。构建的PT-，雷克斯DEST30载体（12301-016 Invitrogen公司）的cDNA使用网关技术（Invitrogen公司）的利益。以cDNA表达的情况下，添加了标签的cDNA将购买方便外源性表达的蛋白质的检测。 </li><li>瞬时转染父母的靶细胞系与pTER或PT-REX的DEST30的质粒（无论是的shRNA表达或cDNA的利益）的转染试剂的选择。的shRNA表达载体的测试，确保可以达到较高的转染效率。 </li><li>收获转染细胞和免疫过程使用相应的抗体，期待您感​​兴趣的基因检测耗尽或过度表达。 </li></ol> 4。与四环素诱导（TET-ON）的shRNA的表达或cDNA使用pTER或PT-REX DEST30载体的的稳定细胞系产生的</p&gt; <ol><li>第1天，种子1×10 6个细胞的&#39;最好&#39;的四环素表达克隆，每10厘米的板使用标准认证的四环素无血清（例如FBS自Invitrogen 16000-014）生长培养基。种子的一个极板与pTER（或的pT-雷克斯DEST30），和一个板作为阴性对照双重选择用于转染。确保尽可能用最低的通道。 </li><li>在第2天，转染用2微克的pTER使用Fugene 6（或的pT-雷克斯DEST30）的一个极板。不要转染第二板。 </li><li>在第3天，添加的标准生长培养基含有5μg/ ml的杀稻瘟素和500微克/毫升吉欧霉素（或1毫克/毫升的G418）。 </li><li>在第4天，细胞分裂，在1/5，1/25，1/50和1/100使用10厘米的板和标准的生长培养基中含有5微克/毫升杀稻瘟素和500微克/毫升吉欧霉素（或1毫克/毫升G418）。准备两个板块每股稀释步骤。 </li><li>每天检查细胞，使确定的第一个星期内的所有单元格未转染的板已经死亡。更改的标准生长介质中含有杀稻瘟和吉欧（G418），每3-4天。 </li><li> 2-3周后，杀稻瘟/吉欧（或blasticidin/G418）的，双抗菌落开始出现。选择板，含有5〜50的菌落挑选单个菌落。 </li><li>当菌落已经达到了一个直径大约5毫米（或更大），使用克隆缸挑选单个菌落。隔离至少24个人的克隆。每个克隆转移到一个单独的24孔组织培养板的孔。 </li><li>文化在含的维护浓度的杀稻瘟素（5微克/毫升），吉欧霉素（250微克/毫升）的克隆[或G418（0.5毫克/毫升）]。当融合时，从每个孔中的细胞分裂成一个单一的孔的6孔组织培养板中。 </li><li>对于每个要测试的克隆，分割1汇合，以及分为三个单独的6孔板的孔中的6孔板中。合流时，冻结细胞从一个势阱。使用剩下的两个井测试四环素，多西环素，米诺环素，罗红霉素，电子诱导表达。添加四环素在一个阱的浓度为1微克/毫升，而离开第二阱四环素免费。 </li><li>经过24至96小时的潜伏期与四环素类，收获细胞和免疫使用相应抗体。较短的四环素孵育时间时，建议的筛选cDNA表达的诱导，而较长的四环素的治疗将是必要的，以确定shRNA介导的耗尽的内源性蛋白质。 </li><li>至少选择两个克隆所需的枯竭/过度表达，并扩大各文化。尽快冻结股票的每一个有前途的克隆。当越来越多的Tet-克隆的实验，始终确保使用的培养基与四环素无血清和维护各自的选择抗生素浓度。 </li></ol>

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没有利益冲突的声明。

我们相信的Tet-系统上，大大方便了对基因功能的分析，特别是在电池系统是难以操纵和/或当操纵基因的细胞的存活是必需的。此外，能够控制基因的表达具有高度特异性春节的系统上提供了一个机会来研究基因功能，在不同的阶段（例如在细胞周期进展或明确定义的分化过程）。这里介绍的方法使研究人员能够生成所需的稳定的Tet-细胞株在合理的时间内（一般为3-6个月，很大程度上取决于四环?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>胎牛血清（FBS） </td><td> Invitrogen公司</td><td> 16000-044 </td><td>测试的春节免费</td></tr><tr><td>杀稻瘟</td><td> Invivogen </td><td>蚂蚁提单-1 </td><td></td></tr><tr><td>吉欧</td><td> Invivogen </td><td>蚂蚁使用-zn-5 </td><td></td></tr><tr><td> G418 </td><td> PAA实验室</td><td> P31-011 </td><td> 100毫克/毫升在媒体</td></tr><tr><td>反，TET02的</td><td> MoBiTec有限公司</td><td> TET02 </td><td>在1/1000至1/2000 WB </td></tr><tr><td> pcDNA6/TR </td><td> Invitrogen公司</td><td> V1025-20 </td><td></td></tr><tr><td> PT-REX DEST30 </td><td> Invitrogen公司</td><td> 12301-016 </td><td></td&gt; </tr><tr><td>四环素</td><td>西格玛</td><td> 87128 </td><td> 2毫克/毫升在乙醇中</td></tr><tr><td>多西环素</td><td>西格玛</td><td> D9891 </td><td> 2毫克/毫升在水中</td></tr><tr><td>克隆缸</td><td> Bellco玻璃公司</td><td> 2090-00808 </td><td>可重复使用的</td></tr></tbody></table>

generation of stable human cell lines with tetracycline-inducible (tet-on) shrna or cdna expression

一个主要的方法在哺乳动物细胞生物学领域是操纵在选定的单元格线的兴趣基因的表达，的目的是显示一个或多个该基因的功能（次）瞬态/稳定过表达或敲除的基因的兴趣。不幸的是，这种做法是不合适的各种细胞生物学研究时，操纵细胞的生长/的增殖缺陷或不需要的细胞分化的基因表达的结果。因此，研究人员已经适应了四环素阻遏蛋白（四环素），采取从E.大肠杆菌四环素抗性操纵子1，生成非常高效和严格的监管系统，以表达在哺乳动物细胞中的cDNA 2,3。总之，四环素 - 已被修改，或者（1）块转录起始的绑定到在Tet-运算符（TO）的启动子区域中添加四环素（称为Tet-off系统）时，或（2）结合的TO在吨他没有四环素（称为的Tet-系统）（ 图1）。鉴于不便Tet-off系统需要四环素（它具有的半衰期为约24小时，在组织细胞培养基）的持续存在的Tet-系统上的已被更广泛地优化，在很紧的发展所导致的和高效的cDNA表达载体系统，在这里使用。 成立后不久的RNA干扰（RNAi）的基因敲除在哺乳动物细胞中，载体表达的短发夹RNA（shRNA片段）进行了描述，非常到的siRNA 5-11类似的功能。然而，这些shRNA介导的敲除的方法具有相同的限制，与传统的基因敲除策略，当靶基因的细胞存活是必不可少的，因为稳定的消耗是不可行的。为了克服这种局限性，面包车韦特灵等 12修改的shRNA表达载体pSUPER 5 </su对&gt;通过插入一个TO中的启动子区域，使他们能够产生稳定的细胞系与四环素诱导耗尽其感兴趣的靶基因。 在这里，我们描述了一个方法，有效地生成稳定的人的春节，可靠地驱动“诱导表达或枯竭，感兴趣的基因的细胞株。使用这种方法，我们已经成功生成春节细胞株，大大促进了分析的的MST / hMOB / NDR级联中心体13,14和凋亡信号15,16。在此报告中，我们描述我们给出的载体，除了描述的两个连续的操作步骤是必要的以有效地产生人的Tet-细胞系（ 图2）。此外，除了概述的产生的一个协议，用于人类的Tet-细胞系中，我们将讨论有关的技术程序和表征的Tet-细胞上的关键方面。

的RPE-1细胞系稳定表达四环素的初始特性的一个例子如图4所示。注意所有的RPE-1克隆表达不同水平的四环素- （比较泳道2和5），而线（作为阴性对照）的亲本细胞不表达外源性四环素蛋白（ 图4，泳道1）。四环素 - 表达RPE-1之间的这种变化的Tet-细胞克隆是意料之中的，因为四环素表达质粒的表达是高度依赖于质粒的整合位点。 表征的稳定的RPE-1的Tet-?...

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

一种快速简便的方法来产生人类细胞系诱导的，可逆的基因表达或shRNA介导的，感兴趣的基因敲除。此方法使研究人员能够可靠地和高再现性操作的细胞系，是很难改变，通过瞬时转染的方法或常规的击倒/基因敲除策略。

（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 ...

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Research

JoVE Journal

Biology

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

35.2K 次观看.  UCL Cancer Institute. 一种快速简便的方法来产生人类细胞系诱导的，可逆的基因表达或shRNA介导的，感兴趣的基因敲除。此方法使研究人员能够可靠地和高再现性操作的细胞系，是很难改变，通过瞬时转染的方法或常规的击倒/基因敲除策略。

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

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

科研

遗传学

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

肿瘤细胞系基因表达的条件性击倒研究单核/巨噬细胞在微环境中的招募

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

癌症研究

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

血管动力尾静脉注射对小鼠肝细胞基因的本构和诱导系统的修饰

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods to investigate the role of key genes involved in cancer cell stemness. Gastric cancer (GC) is one of the most common and mortal types of cancers. Gastric cancer stem cells (GCSCs) are thought to be the root of gastric cancer relapse, metastasis and drug resistance. Understanding GCSCs&#160;biology is needed to advance the development of targeted therapies and eventually to reduce mortality among patients. In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

结合条件敲击和适应球形成测定研究患者衍生胃癌干细胞的干细胞相关基因

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...

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 ...

免疫与感染

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in C2C12 myoblasts can be applied to study the role of these proteins in skeletal muscle development. Here, we describe a protocol to deplete the ECM protein ADAMTSL2 as an example, using small-hairpin (sh) RNA in C2C12 cells. Following transfection of shRNA plasmids, stable cells were batch-selected using puromycin. We further describe the maintenance of these cell lines and the phenotypic analysis via mRNA expression, protein expression, and C2C12 differentiation. The advantages of the method are the relatively fast generation of stable C2C12 knockdown cells and the reliable differentiation of C2C12 cells into multinucleated myotubes upon depletion of serum in the cell culture medium. Differentiation of C2C12 cells can be monitored by bright field microscopy and by measuring the expression levels of canonical marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC) indicating the progression of C2C12 myoblast differentiation into myotubes. In contrast to the transient knockdown of genes with small-interfering (si) RNA, genes that are expressed later during C2C12 differentiation or during myotube maturation can be targeted more efficiently by generating C2C12 cells that stably express shRNA. Limitations of the method are a variability in the knockdown efficiencies, depending on the specific shRNA that may be overcome by using gene knockout strategies based on CRISPR/Cas9, as well as potential off-target effects of the shRNA that should be considered.

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin shRNA

We provide a protocol to stably knock down genes encoding extracellular matrix (ECM) proteins in C2C12 myoblasts using small-hairpin (sh) RNA. Targeting ADAMTSL2 as an example, we describe the methods for the validation of the knockdown efficiency on the mRNA, protein, and cellular level during C2C12 myoblast to myotube differentiation.

使用小毛针（sh）RNA在C2C12霉细胞细胞系中编码细胞外基质蛋白的基因稳定击倒

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

发电<em&gt;德诺</em&gt;抗原特异性人T细胞受体通过中心的Hemichain的逆转录病毒转导

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

E4orf4诱导的细胞死亡的击倒效果的基因表达和测量条件击倒细胞系的制备

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various ...

生物学

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

另类文化的人多能干细胞的生产，维护和遗传分析

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Short Hairpin RNA-Mediated Gene Knockdown in iHSPCs In Vitro: A Lentivirus-Based shRNA Expression System Delivery into iHSPCs for Knockdown of Specific Gene Expression

Source: Hsiao, Y. L., et al. <a href="https://www.jove.com/v/62730">Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro.</a> J. Vis. Exp. (2022).
In this video, we demonstrate a method to perform transduction of shRNA lentiviral vectors to obtain stable knockdown cell lines in immortalized hematopoietic stem and progenitor cells (iHSPCs).

Short Hairpin RNA Mediated Gene Knockdown in iHSPCs In Vitro: A Lentivirus-Based ShRNA Expression System Delivery into iHSPCs for Knockdown of Specific Gene Expression and Generation of Stable Knockdo

体外 iHSPC 中的短发夹 RNA 介导的基因敲低:一种基于慢病毒的 shRNA 表达系统递送到 iHSPC 中以敲低特异性基因表达

Source: Hsiao, Y. L., et al.  Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell ...