使命是Sigma - Aldrich公司的生物技术LP的注册商标。 LentiPlex是Sigma - Aldrich公司的生物技术LP的商标。

LentiPlex汇集屏幕设置要确定利益的shRNA序列，这是关键的设置屏幕等，多数细胞接收只有一个shRNA的构建。通过使用一个低MOI（感染复数），每个单元的多个integrants的概率大大下降。然而，转导效率，并因此所需的水分，在很大程度上取决于靶细胞类型。因此，当务之急是确定的最佳MOI进行前开始在一个新的细胞类型的屏幕。 1。转导靶细胞团LentiPlex汇集shRNA文库<ol><li>实际的教学语言使用，会因不同类型的细胞。如果复制是所需的板数必须相应增加。此外，通常不会有利于结合不同子池。保持的子池的独立将有助于在反卷积过程。确保propeř每个子池的数量，在传导过程中应用的组织培养皿中的标签。低MOI建议，以减少每个细胞多个integrants的可能性。 </li><li>第1天 - 种子60毫米的菜肴适当数量足够的细胞获得〜70％汇合于翌日（共10池，执行的每个= 30种共一式三份）。对于我们的研究中，60 mm培养皿接种6 × 105 A549细胞，实现了70％的汇合前向传导。这可确保细胞仍然在指数生长期。增长率将有所不同的细胞类型和屏幕开始之前，必须先确定。 </li><li>让细胞在37℃孵育过夜，在培养箱中5％的CO 2的气氛彗星坚持。 </li><li> （可选）包含两个额外的60毫米的菜肴与阴性对照的shRNA转。标签菜“控制”SHC001H和“控制”为SHC002H的。 </li><li>第2天 - 厚膜积体电路次Ë细胞与病毒颗粒LentiPlex。转天，在冰上解冻慢病毒颗粒。 </li><li>解冻颗粒传输层流罩，并保持在冰上，如果不立即使用。 </li><li>计算多少每一个人的病毒子池添加到细胞，所有细胞培养皿MOI 1。 <ol><li> 范例：1 × 10 6细胞/皿;病毒滴度= 5 × 10 8 TU /毫升;所需的MOI 1。一）1 × 10 6细胞/皿X（MOI 1）= 1 × 10 6转导单位（TU）的需要二）1 × 10 6（译文）/（5.0 × 10 8 TU /毫升）从C = 2μL慢病毒原液应加适当的菜。稀释病毒的计算量在100-300μL完整的媒体，以确保更好地添加到细胞培养皿时，病毒的分布。 </li></ol></li><li>前种子细胞取出介质。添加完整的媒体conta状态寄存器hexadimethrine溴铵溶液，每道菜。媒体hexadimethrine溴化建议终浓度为8微克/毫升。如果你发现它是有毒的靶细胞，使用较少的hexadimethrine溴化。 </li><li> shRNA慢病毒颗粒添加适当的菜，在按照预定的实验设计。轻轻旋流板均匀地分布在细胞的病毒。 </li><li>孵育18-20小时，在37 °培养箱在5％的CO 2的气氛中的C。 </li><li> （可选）重复3-8 SHC001H相同MOI在控制盘（空载体的shRNA控制）A和SHC002H（炒的shRNA控制）控制盘B.相同对待这些菜在整个实验的实验菜。 </li><li>第3天 - 更改媒体。吸含病毒的媒体和补充新鲜完整的媒体（不包括hexadimethrine溴化）。在37 ° C过夜培养细胞。 </li></ol> 2。治疗镀细胞治疗组件/试剂<ol><li>第4天 - 从油井吸媒体，并添加含有最佳浓度治疗组件的新媒体。至于与培养条件下，治疗会有所不同，事先应确定适当的浓度和持续时间。在我们的研究中，我们使用100纳克/毫升TRAIL和紫杉醇1微米。细胞经48小时。 </li><li>存活细胞应重新接种到T75烧瓶，并允许扩大到近100％汇合。 </li><li> （可选）在选拔结束，检查控制盘和控制盘二盘A和Dish B，每个人都有至少一个汇集库菜少殖民地。如果有意外的高盘数菌落，比传导过程已经影响到相关所需的表型或选择性压力的一个途径，是没有足够强大的检测和重新优化可能需要。如果碟B包含了太多的殖民地，然后表达shRNA的RNAi途径参与可能有利益的表型的影响。如果这是与SHC001H重复，以确保效果是不特定SHC002H的。 </li></ol> 3。多克隆细胞群从一个解卷积<ol><li>收获从每个子池细胞的异质人口和分离基因组DNA。在我们的试验中，细胞洗涤简要的PBS和胰酶消化前基因组DNA的提取使用GenElute哺乳动物的基因组小量制备试剂盒和供应协议（Sigma - Aldrich公司，G1N70）。另外，任何其他的基因组纯化试剂盒，目前市场上可用于DNA分离。重要的是要培养细胞的起点金额按照协议的建议。 </li><li> PCR扩增的目标。 shRNA的模板恢复过程，使扩增从选定的细胞群的shRNA插入全池，或indivi检索双shRNA的模板，从殖民地，被单独挑选和扩大。扩增的shRNA插入控制和选定的靶细胞后，PCR产物测序。 </li><li>此PCR扩增步骤进行了优化，使用预拌西格玛，产品编号P2893。其他试剂可用于放大，但可能需要修改成功扩增循环条件和/或镁离子浓度。扩增引物浓​​度为20毫米，包括与LentiPlex套件。 </li><li>设置如表1所述的PCR反应。列出的顺序添加组件。所述的金额为50μL反应。对于更多的反应，反应所需的数字规模主结构组件，其中包括10％盘盈。建议的DNA模板量为50-100毫微克。 </li><li>强烈建议一个反应模板组成的人类使命的shRNA阳性对照稀释的appropr矢量iate浓度。与此模板的成功放大，表明PCR检测组件和循环条件是足够的。注意：为了防止交叉污染的实验样品和试剂的污染，因此建议，在随后的PCR反应是成立一个单独的位置稀释阳性对照。 </li><li>保存表2中所列的PCR程序到您的热循环。 </li><li>对于每个样品，结合3μLPCR反应1μL染料和electrophorese沿着5μLDirectLoad™PCR扩增100 bp的低梯，在1％琼脂糖凝胶产品编号D3687确认存在一个309 bp的扩增。 </li><li> PCR扩增：PCR产物亚克隆TOPO克隆使用的TOPO - TA克隆试剂盒，按照制造商的协议（Invitrogen公司，K4575 - 01）。其结果是，在每个向量中一个PCR产物。 </li><li> 250个人（每片含个体的PCR扩增）的细菌菌落进行分离和成长N在2毫升100毫克/ mL氨苄青霉素的LB培养。质粒DNA的提取，使用GenElute质粒小量制备试剂盒（Sigma - Aldrich公司，PLN70）。 </li><li>纯化的质粒DNA用EcoRI消化和电泳确认插入的存在。 </li><li>质粒DNA样本，然后测序，使用的LentiPlex shRNA序列搜寻资料库与LentiPlex库提供的shRNA插入确定。 </li></ol> 4。目标识别和验证<ol><li> shRNA序列5&#39; - GAAACACCGG - 3&#39;启动。输入至少在未来10进入封闭的shRNA序列搜索访问数据库形式的搜索框中的查询序列，但不少于21个核苷酸。 </li><li>选择汇集的shRNA物种。也可以选择从该序列被发现的子池。 </li><li>现在点击“寻找潜在的点击”按钮进行搜索。这应确定相应的TRC的shRNA序列（S）比赛日Ë测序数据。 TRC的shRNA序列的更多信息（S）和相应的靶基因可以很容易地发现：Sigma - Aldrich公司您最喜爱<a href="http://www.sigma.com/yfg">的</a>基因http://www.sigma.com/yfg </li><li>应确定的目标基因与原来的真相与和解委员会的克隆加上至少有两个额外的shRNA的目标是相同的基因1 shRNA的1以及格式重复原筛查检测验证。真正的点击将显示在相同的表型多数水井。 </li></ol> 5。代表性的成果一个LentiPlex汇集屏幕工作流程的一个例子是详细的图1。转导，细胞增殖TRAIL和紫杉醇的存在被允许扩大，直到瓶融合。收获和受到的PCR扩增和克隆基因组DNA，如图1所示，被提交测序和shRNA鉴定之前在Figu重1 C. 250个克隆进行测序分析，这25多个克隆代表其余225名代表只有1克隆，并没有在这个时候推行。 正如图2所示的几个候选基因，其中包括TBX3，PPP2CA，AKT2派代表出席了由多个克隆。 TBX3的T - box转录因子，共有4个克隆出现，已在肿瘤迁移 5牵连。已被证明PPP2CA下调，减轻雄激素剥夺诱导细胞周期阻滞和防止细胞凋亡 6，以维持在雄激素缺乏的条件下培养的LNCaP细胞的生长。 AKT2是一个RAC -β的丝氨酸/苏氨酸蛋白激酶和公认的癌基因，证明扮演几个角色，在癌症发展的七，八。 <img src="/files/ftp_upload/3305/3305table1.jpg" alt="表1" /> *终浓度的Mg 2 +溶液中，将3毫米;从Taq酶预拌氯化镁溶液和1.5毫米1.5毫米贡献。 表1。Lentiplex PCR反应条件 <img src="/files/ftp_upload/3305/3305table2.jpg" alt="表2" /> 表2。Lentiplex PCR扩增程序 <img src="/files/ftp_upload/3305/3305fig1a.jpg" alt="图1a" /> <img src="/files/ftp_upload/3305/3305fig1b.jpg" alt="图1b" /> <img src="/files/ftp_upload/3305/3305fig1c.jpg" alt="图1c" /> 图1。 （一）汇集LentiPlex屏幕，（二）多克隆反褶积的工作流程，和（C）的shRNA目标的识别 。 A. LentiPlex汇集屏幕首先，种子细胞，然后加入shRNA的子池（共10池，各一式三份进行，共30菜）。其次，对待治疗组件/试剂（在我们的例子中，TRAIL和PAclitaxel，分别）。然后，补种在瓶中存活细胞，并允许扩大。收获异构人口细胞从每个子池和隔离基因组DNA。 B.多克隆反卷积执行 PCR扩增的DNA含有的shRNA插入。因为模板基因组DNA也多克隆的PCR产物是统一的大小，但在序列多克隆。 PCR产物克隆到载体，然后转化成主管细菌。 C.鉴定shRNA的目标是孤立的单个菌落和质粒DNA提取。每个克隆包含一个单独的PCR片段的克隆载体。质粒DNA消化确认存在插入和测序。 shRNA的插入是确定使用LentiPlex shRNA序列搜索数据库。 <img src="/files/ftp_upload/3305/3305fig2.jpg" alt="图2" /> 图2。鉴定可didate基因的25个候选基因进行鉴定，有多个点击。 225个候选基因只有1命中，而无法追查。请每个候选基因的单个克隆细目见补充表1。 <img src="/files/ftp_upload/3305/3305suptable1.jpg" alt="表1" /> 补充表1。真相与和解委员会的图书馆个人从多克隆测序基因ID中的克隆点击检出克隆 。

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马修j Coussens，康特尼Corman，阿什利L.菲舍尔，杰克西米和约翰Swarthout受雇于Sigma - Aldrich公司，这使得工具和本文中使用的试剂。

在这项研究中，我们提出一个全基因组的屏幕，以确定参与紫杉醇/ TRAIL的治疗后的A549细胞的细胞毒作用的基因。在全基因组的屏幕汇集方法的使用减少的时间，成本和设备投资通常与传统的摆着屏幕关联。屏幕成功的关键是事先进行优化实验。传导条件和教学语言必须凭经验确定。 选择方法应允许强大的选择显示所需的表型（如生存，表面蛋白的表达，等）的细胞。这些参数的优化将降低检测的误报数量。 在这里，基因组DNA被收获从选定的单元格批量人口和TOPO克隆识别单个的shRNA插入。这个实验的一个可能的改进已选择活细胞，使用流式细胞仪，以确保只有活细胞被收集。在筛选实验的具体目标的shRNA转导所确定的目标将被验证。真正的点击将显示在大多数水井的表型。 LentiPlex汇集库及其应用灵活。它是兼容的下游应用广泛，可用于积极或消极的选择策略。根据筛选条件的shRNA插入的反卷积是通过PCR /克隆，基因芯片，或新一代测序9，10，虽然这些技术的力量和速度deconvolute的RNAi屏幕已经非常成熟，与芯片的关注和完成。新一代测序方法是生物信息资源和知识，需要正确地分析和解释实验结果的可用性。与这些技术相关的成本，可以经常进行全基因组屏幕的一个障碍。 APCR /克隆为基础的方法，而不是如芯片和新一代测序强大，有效和成本更低的替代方案。 安捷伦现在提供了一个自定义的数组的基础上TRC shRNA文库LentiPlex屏幕提供进一步的援助。这些选项允许研究人员使用LentiPlex研究细胞功能的多元化。

<html><table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 产品编号 </td></tr><tr><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=SHPH01%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">LentiPlex汇集阵列</a> </td><td> <a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">Sigma - Aldrich公司</a> </td><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=SHPH01%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">SHPH01</a> </td></tr><tr><td> A549细胞</td><td> ATCC </td><td> CCL - 185 </td></tr><tr><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=T5694%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">TRAIL</a> </td><td> <a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">Sigma - Aldrich公司</a> </td><td><html> oductDetail.do？LANG = EN＆N4 = T5694％7CSIGMA＆N5的= SEARCH_CONCAT_PNO％7CBRAND_KEY及F =规格“&gt; T5694 </td></tr><tr><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=T7402%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">紫杉醇</a> </td><td> <a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">Sigma - Aldrich公司</a> </td><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=T7402%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">T7402</a> </td></tr><tr><td>地形助教克隆试剂盒</td><td> Invitrogen公司</td><td> K4575 - 01 </td></tr><tr><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=P2893%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">订JumpStart的Taq预拌</a> </td><td> <a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">Sigma - Aldrich公司</a> </td><td>ONCAT_PNO％7CBRAND_KEY＆F =规范“&gt; P2893 </td></tr><tr><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=PLN70%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">GenElute质粒小量制备试剂盒</a> </td><td> <a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">Sigma - Aldrich公司</a> </td><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=PLN70%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">PLN70</a> </td></tr><tr><td> EcoR1 </td><td> New England Biolabs公司</td><td> R0101 </td></tr><tr><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=H9268%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">hexadimethrine溴</a> </td><td> <a href="http://www.sigmaaldrich.com/united-states.html" target="_blank">Sigma - Aldrich公司</a> </td><td> <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=H9268%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">H9268</a> &lt;/ TD&gt; </tr></tbody></table>

mission lentiplex pooled shrna library screening in mammalian cells

RNA干扰（RNAi）是一种内在的细胞基因表达调控机制。利用这一制度的先天力量，使我们能够击倒基因表达水平的基因功能研究的损失。 执行RNA干扰主要有两种方法。首先是利用小分子干扰核糖核酸（siRNAs）的化学合成，第二个利用短发夹核糖核酸（的shRNA）质粒1编码内。后者可直接进入细胞转染或包装成复制不称职的慢病毒颗粒。使用慢病毒的shRNA的主要优点是引入了多种类型的细胞，他们有能力稳定地融入了长期的基因敲除和选择的基因组，其疗效进行高通量功能屏幕的损失减轻。为推动这项工作，我们已经创建了汇集的shRNA库LentiPlex。 管理信息系统SION LentiPlex人类的shRNA汇集图书馆是一个全基因组慢病毒池生产使用一种专有工艺。该库包含超过75,000的shRNA结构针对15000的真相与和解委员会收集+人类基因2。每个库产品发布前进行测试，以确保稳健的库覆盖shRNA的代表性。图书馆是提供在至少5 × 10 8 TU /毫升通过P24检测滴度的准备使用的慢病毒格式和预分为十子池约8,000 shRNA的构造每个。还提供了扩增和测序引物为下游目标识别。 以往的研究，建立了对TRAIL协同抗肿瘤活性与紫杉醇相结合时，A549细胞，人类肺癌细胞株 3 ，4。在这项研究中，我们展示了汇集LentiPlex shRNA文库的应用迅速进行有关的基因在细胞毒性Ø积极选择屏幕f A549细胞时，接触到TRAIL和紫杉醇。与高通量筛选经常遇到的障碍之一是在反褶积的成本和困难，我们也详细的成本效益的多克隆利用传统测序方法。

Watch this Scientific Journal Video about MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells at JoVE.com

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

在这里，我们使用人类LentiPlex汇集库和传统的测序方法，以确定靶基因，促进细胞的存活。我们演示了如何设置和deconvolute一个LentiPlex屏幕和验证的结果。

特派团LentiPlex汇集在哺乳动物细胞中的shRNA文库筛选

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

mission-lentiplex-pooled-shrna-library-screening-in-mammalian-cells

Research

JoVE Journal

Biology

18.5K 次观看.  Sigma-Aldrich. 在这里，我们使用人类LentiPlex汇集库和传统的测序方法，以确定靶基因，促进细胞的存活。我们演示了如何设置和deconvolute一个LentiPlex屏幕和验证的结果。

视频: MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing和传代的哺乳动物细胞

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

科研

生物学

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

miRNA的目标使用的任务目标ID库的全基因组屏幕

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

在培养的哺乳动物细胞大规模并行报告基因分析

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

定向进化法<em&gt;酿酒酵母</em&gt;：突变体库的创建和筛选

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...