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-?...

Watch this Scientific Journal Video about Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression at JoVE.com

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 ...

generation-stable-human-cell-lines-with-tetracycline-inducible-tet-on

Research

JoVE Journal

Biology

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

35.0K 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

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

人类胚胎干细胞传代的色调线与胰蛋白酶

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

科研

生物学

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

人CD40激活的B细胞的生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

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 ...

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.