Name: silencing of brca2 to identify novel brca2-regulated biological functions in cultured human cells
Uploaded: 2015-08-12T14:00:00.000+00:00
Duration: 9 min 24 s
Description: Watch this Scientific Journal Video about Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells at JoVE.com

This work was supported by FIRB-Merit grants RBNE08YFN3_005 and RBNE08HWLZ_012, and the Italian Ministry of Economy and Finance to the CNR for the Project &#8220;FaReBio di Qualit&#224;&#8221;.

1.准备的siRNA的解决方案<ol><li>重悬5纳摩尔BRCA2基因的siRNA干燥粒料加扰（Ctrl键）和5纳摩尔在100μl无RNase水（最终的siRNA浓度为50μM）。这是所述siRNA的库存，并且必须被储存在-20℃于10微升等分试样。 </li><li>取从所述siRNA库存10微升等分试样并加入无RNase水40微升，得到10μM的siRNA的工作溶液。该稀释必须存储在-20℃直到使用。 注:siRNA的工作溶液不应经受冷冻/解冻的3倍以上。 </li></ol> 2.播种人表皮细胞系<ol><li>在37℃，5％的CO 2从人上皮细胞系生长的细胞单层在组织培养板在培养箱中生长培养基中。 </li><li>洗细胞在板一次，用RT的PBS（10毫升的PBS为100毫米板）。 </li><li>以从板取下细胞，加入2ml的0.25％胰蛋白酶的/ 0.53毫摩尔EDTA溶液孵育3-5分钟取决于细胞类型。 </li><li>之前收集分离的细胞中，加入2ml含10％胎牛血清的完全培养基的中和胰蛋白酶。 </li><li>转移细胞到15毫升管。 </li><li>在吊桶式转子离心细胞在320-330×g离心3分钟，在4℃。 </li><li>重悬细胞沉淀在5ml抗生素的培养基中。 </li><li>算在Burker室中的显微镜下或通过根据制造商的说明使用自动计数系统的细胞。 </li><li>种子细胞在6孔板24小时（0.5-1×10 6个细胞在2ml培养基的每孔）后为约80％汇合。最佳细胞数接种可以取决于特定细胞系和生长速度各不相同。 注意:不要在中在这一点上添加抗生素，因为抗生素会负transfe的干扰效率ction。这是很重要的，该细胞是在低培养传代（&lt;10），以实现BRCA2 siRNA转染的高效率。 </li></ol> 3.转染细胞的siRNA <ol><li>电镀细胞后二十四小时内，继续进行第一轮的转染。 <ol><li>稀释6微升基于脂质的10特异性的siRNA转染试剂在130微升减少血清培养基。 </li><li>在130微升减少血清培养基稀释3微升10μM的siRNA（30皮摩尔）。 </li><li>结合稀释的siRNA与稀释的转染试剂，孵育在室温5-10分钟。 </li><li>在温育后，除去从细胞培养基中，并添加新鲜培养基预先加热至37℃（无抗生素）2毫升</li><li>加入250微升的siRNA转染试剂复合物的细胞（该量是用于一个6孔板的单个孔）。 </li><li>孵育细胞在加湿培养箱中在3776下用5％的CO 2。 </li></ol></li><li> 24小时后，继续进行第二轮的转染重复步骤3.1.1-3.1.5并作以下修改为3.1.2步:稀释5微升10μM的siRNA（50皮摩尔）的130微升减少血清培养基。 <ol><li>分析前孵育细胞1-2天，在37℃。 注:两轮转染和使用在第二轮高的siRNA /转染试剂的比例是必不可少得到BRCA2沉默的效率高。 </li></ol></li></ol> 4.裂解细胞免疫分析<ol><li>制备新鲜的裂解缓冲液与含有非离子型，非变性去污剂辛基苯氧基的1％，5毫焦磷酸钠，1mM的原钒酸钠，10mM的氟化钠，2mM的苯基甲基磺酰氟，10微克的PBS（pH为7.4）/ ml亮肽素，10微克/ ml抑肽酶。保持它在冰上。 </li><li>除去从细胞以及w的介质用冷PBS（2毫升/孔）在该板一次灰它们。为了防止交叉污染的PBS，它完全从板块中删除。 </li><li>加入200μl裂解缓冲液到每个孔中。 注意:如果该细胞必须也用于其他测定法，trypsinize细胞如步骤2.1-2.6中描述和悬浮细胞中根据应用适当的缓冲/介质中使用。 </li><li>轻轻转动该板均匀地分布在裂解缓冲液孵育15分钟，在4℃下在旋转器上。 </li><li>收集在使用细胞刮刀冰离心管的细胞裂解物。 </li><li>离心机在17900×g离心25分钟，在4℃，并收集在一个新的管中的上清液。 </li><li>测量使用根据制造商的说明市售蛋白质测定蛋白质浓度。 </li></ol> 5. BRCA2免疫印迹分析<ol><li>通过稀释加入50ml 20×Tris-醋酸SDS电泳b制备运行缓冲uffer（50毫曲辛，的50mM Tris碱，0.1％SDS，pH值8.24）950毫升蒸馏水。 </li><li>制备的样品如下:添加15微克总细胞裂解物，将5μl的4倍样品缓冲液含有十二烷基硫酸锂，在pH 8.4，加入2μl10×样本还原剂（0.5M DTT），和裂解缓冲液至20中的最终体积微升。 注意:此协议允许检测BRCA2的正常细胞系也当使用总蛋白的较低量（降至5微克）。 </li><li>变性的样品在55℃下进行10分钟。 注:关键是要使用55℃。因为BRCA2蛋白是热敏性11，更高的变性温度导致完整的全长（390 kDa）的BRCA2蛋白一致损失。 </li><li>根据制造商的说明建立的电泳室。 </li><li>装载样品和10微升的高分子量预染色的蛋白质标准进行预制凝胶（Tris-醋酸3-8％），并在运行120伏的AB出2小时12。停止55 kDa的蓝色标记离开预制胶前的电泳运行。 </li><li>准备转移缓冲液用50毫升20×转移缓冲液，加入100毫升100％甲醇和850毫升水中。 </li><li>激活PVDF膜（0.45微米孔径）通过浸泡在100％甲醇5分钟，用蒸馏水漂洗并平衡在转移缓冲液至少5分钟。浸泡在转移缓冲液传送装置的海绵，在4℃直至使用。 </li><li>当电泳运行结束，组装在以下顺序（自底向上）传输堆栈夹层:在转移缓冲液饱和3海绵一3MM级纸张饱和在转移缓冲液，凝胶，活化PVDF膜，人们3MM在转移缓冲液饱和级纸，三海绵饱和传输缓冲区13,14。放置在栈入装置和转移蛋白在350mA 4小时，在4℃或在180毫安过夜，在4℃。 注意:存在BRCA2一个非常大的蛋白质，它是非常重要的，从1小时延长转印时间（所建议的制造商，大多数传输协议），以4小时或过夜，以确保高的分子量的蛋白质的完整的传输。另外，由于长期的传输时间，有必要在4℃下进行杂交在冷室中，以尽量减少过热和促进三明治拆卸。半干转移系统是不利于传输大量的蛋白质。 </li><li>转移后，用TBS洗膜，阻止它为1小时，在RT在TBS-吐温0.25％（TBS-T）含有5％脱脂奶粉和探针过夜，在4℃下用30微升抗BRCA2兔多克隆抗体（ 200微克/毫升），稀释于9ml TBS-T + 5％脱脂奶粉[1:300（体积/体积）稀释。 </li><li>用TBS-T洗膜三次（每次10分钟），然后孵育的过滤器1小时，用1微升辣根过氧化物酶缀合的抗兔次级抗体稀释在10的TBS毫升-T + 5％脱脂奶粉。此后，用TBS-T洗膜三次（每次10分钟）。 </li><li>通过ECL进行免疫检测使用增强的化学发光（ECL）的Western印迹检测试剂，按照制造商的说明来揭示BRCA2蛋白。可视化高分辨率ECL电影immunochemiluminescent带。 </li><li>在10 1000（10微升抗微管蛋白抗体的制备:与抗体通过使用单克隆抗体与β微管蛋白稀释在1执行免疫检测对于持家基因，如β微管蛋白（55 kDa）的，在相同的过滤器作为负载对照毫升TBS-T + 5％无脂干牛奶）中1小时，在室温。洗和孵化如对于BRCA2（5.10-5.11）第二抗体，除了使用辣根过氧化物酶缀合的抗小鼠代替抗兔二级抗体。 </li><li>获得与immunochemiluminescent频带印象的膜的数字图像并分析由专用的图像分析软件的谱带强度（ 如，ImageJ的）。 </li></ol>

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The authors have nothing to disclose.

由于BRCA2基因导致几个癌症类型，包括女性和男性乳腺癌，卵巢癌，前列腺癌，胰腺癌和黑素瘤3,4-风险增加的种系突变，一些研究已经进行了解的生物学功能在BRCA2蛋白质。大部分这些研究是基于基因主要是由于在分析像BRCA2巨蛋白的技术困难。此处描述的用于沉默和分析BRCA2蛋白表达的方法，提供了有效的工具来调查BRCA2肿瘤抑制基因的功能的生物测定的广谱。沉默协议是基...

在BRCA2（乳房癌易感基因-2）基因编码肿瘤抑制蛋白，发挥在通过调节重组酶的Rad51 1的功能修复DNA双链断裂了至关重要的作用。 BRCA2也被牵连于转录的调节和细胞周期调控2。在BRCA2基因的种系突变诱导的常染色体显性遗传易感性乳腺癌和在妇女卵巢癌和前列腺癌的男性，以及其他类型的癌症3,4-倾向。然而，尽管在发展癌症，BRCA2基因突变是抑制或减少BRCA2功能可能使肿瘤细胞更容易受到化疗剂引起的DNA损伤5-7的风险增加。零星的癌症表现出BRCA2基因突变（&lt;3％），尽管BRCA2蛋白水平降低已在一些癌症类型被检测到低速率，这表明BRCA2蛋白通过非突变依赖性机制-7,8-肿瘤发生过程中可能会丢失在零星的癌症。因此，要充分认识BRCA2功能在癌症生物学的上下文中以及在其他生物的设置是重​​要的。 用于鉴定在哺乳动物细胞中的基因的新颖功能的强大的工具是沉默其表达。作为一个例子，在各种正常上皮细胞的沉默BRCA2的表达最近导致了新颖BRCA2作为失巢凋亡抗性，采集的癌细胞侵入和转移能力9中的一个重要步骤调 ​​节器的功能的识别。基因沉默可以通过引入细胞中任一小干扰（SI）RNA或短发夹（sh的）RNA分子靶向特定基因来实现。 siRNA的高效力和它的易用性使得它的优先工具沉默实验旨在获得新的见解关键的生物进程第二，以确定新的治疗目标。然而，基因表达的有效的敲低可能并不容易实现对所有的基因，可能是高度可变的取决于细胞类型。因为在细胞内蛋白质水平的降低是所研究的最相关的表型，这是至关重要的免疫印迹的基因产物的分析以量化基因沉默。与这方面，BRCA2蛋白呈现了进一步的挑战:作为一个大的蛋白质（约390 kDa的），技术困难确实存在对常规的生化分析，包括免疫印迹。 我们在这里报告BRCA2在人类上皮细胞系的高效沉默和迅速和成功的检测用免疫印迹法击倒BRCA2蛋白优化的协议。

<table><tbody><tr><td>BRCA2 siRNA</td><td>Dharmacon</td><td>L-003462-00</td><td>SMARTpool: ON-TARGETplus BRCA2 siRNA</td></tr><tr><td>Ctrl siRNA</td><td>Dharmacon</td><td>&amp;nbsp;D-001810-10-05</td><td>ON-TARGETplus Non-targeting Pool</td></tr><tr><td>Lipofectamine RNAiMAX Reagent&amp;nbsp;</td><td>Life Technologies</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/13778075&quot;&gt;13778075&lt;/a&gt;</td><td>Transfection Reagent</td></tr><tr><td>OPTI-MEM I Reduced Serum Medium</td><td>Life Technologies</td><td>31985-070</td><td>Medium for transfection procedure</td></tr><tr><td>NuPAGE Tris-Acetate 3-8% 1,5 mm Gel precast</td><td>Life Technologies</td><td>EA0378</td><td>Gel for protein electrophoresis</td></tr><tr><td>NuPAGE LDS Sample buffer&amp;nbsp;</td><td>Life Technologies</td><td>NP0007</td><td>Reagent for protein electrophoresis</td></tr><tr><td>NuPAGE Sample Reducing agent (10x)</td><td>Life Technologies</td><td>NP0004</td><td>Reagent for protein electrophoresis</td></tr><tr><td>NuPAGE Antioxidant</td><td>Life Technologies</td><td>NP0005</td><td>Reagent for protein electrophoresis</td></tr><tr><td>HiMark Pre-stained protein standard</td><td>Life Technologies</td><td>LC5699</td><td>Prestained marker for gel electrophoresis</td></tr><tr><td>XCell SureLoc Mini-Cell&amp;nbsp; System</td><td>Life Technologies</td><td>EI0002</td><td>Equipment for protein electrophoresis/transfer</td></tr><tr><td>NuPAGE Tris-Acetate SDS Running Buffer</td><td>Life Technologies</td><td>LA0041</td><td>Reagent for Western blotting</td></tr><tr><td>PVDF membrane</td><td>Millipore</td><td>IPVH00010</td><td>Reagent for Western blotting</td></tr><tr><td>NuPAGE Transfer Buffer</td><td>Life Technologies</td><td>NP0006-1</td><td>Reagent for Western blotting</td></tr><tr><td>BRCA2 H-300 rabbit polyclonal antibody</td><td>Santa Cruz Biotechnology</td><td>sc-8326</td><td>1:300 dilution</td></tr><tr><td>Beta-tubulin monoclonal antibody</td><td>Sigma Aldrich</td><td>T4026-100UL</td><td>1:1,000 dilution</td></tr><tr><td>Skim Milk powder</td><td>Sigma Aldrich</td><td>70166</td><td>Reagent for Western blotting</td></tr><tr><td>Tween-20</td><td>Sigma Aldrich</td><td>P1379</td><td>Reagent for Western blotting</td></tr><tr><td>SuperSignal West Pico/Femto Chemiluminescent Substrate</td><td>Pierce Thermo Scientific</td><td>34080/34096</td><td>Chemiluminescence system</td></tr><tr><td>PNT1A cells</td><td>SIGMA Aldrich (for ECACC)</td><td>95012614</td><td>PNT1A human, normal prostate epithelium immortalized with SV40</td></tr><tr><td>Nthy cells</td><td>SIGMA Aldrich (for ECACC)</td><td>90011609</td><td>Nthy-ori 3-1 Cell Line human, thyroid follicular epithelial cells</td></tr><tr><td>Cell Proliferation Kit I (MTT)</td><td>Roche</td><td>11465007001</td><td>Kit for cell proliferation assays</td></tr></tbody></table>

silencing of brca2 to identify novel brca2-regulated biological functions in cultured human cells

肿瘤抑制蛋白BRCA2及其检测常规生化分析的沉默代表了由于人类BRCA2蛋白（约390 kDa的）的大尺寸巨大的技术挑战。我们报告的标准siRNA转染和免疫印迹协议的修改来沉默人类BRCA2和检测内源性BRCA2蛋白，分别在人上皮细胞系。关键的步骤包括一个高的siRNA转染试剂的比例和相同的实验中的两个随后的几轮siRNA转染的。使用这些和其他的修改标准协议中，我们一致地实现人类BRCA2基因的70％以上的沉默作为判断免疫印迹分析用抗BRCA2抗体。此外，细胞裂解物在55℃，而不是传统的70-100℃，并在免疫印迹程序的其他技术的优化变性允许检测完好BRCA2蛋白偶的wh烯极低量原料可用或当BRCA2蛋白质表达水平非常低。 BRCA2的人类细胞中沉默效率提供了一个有价值的策略，以扰乱BRCA2功能的细胞中具有完整BRCA2，包括肿瘤细胞，以检查新的分子途径，并且可能会受致病BRCA2基因突变的肿瘤中的细胞功能。此协议为有效的沉默等"大"蛋白如BRCA2的分析，适应应该是很容易实现的。

在继续进行的生物/生化测定来检查BRCA2沉默对细胞功能的影响，第一个步骤是通过使用一个加扰的siRNA（非靶向siRNA）并排BRCA2的siRNA（ 图1A确认沉默的特异性）。它执行第二轮的siRNA转染的具有高的siRNA /转染率， ​​以得到BRCA2沉默的高效率是非常重要的。实际上，仅执行一个回合siRNA转染的或用在第二轮（25皮摩尔的siRNA和6微升转染试剂）低级的siRNA转染的比例会导致低...

Watch this Scientific Journal Video about Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells at JoVE.com

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

沉默<em&gt; BRCA2</em&gt;以确定新的BRCA2调控的生物学功能在人体细胞培养

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

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Research

JoVE Journal

Biology

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

9.0K 次观看.  National Research Council. Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented. 

视频: Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

科研

癌症研究

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C&#62;T, c.154C&#62;T, c.3847C&#62;T, c.5056C&#62;T, and c.4986+5G&#62;A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

People with BRCA1 mutations have a higher risk of developing cancer, which warrants accurate evaluation of the function of BRCA1 variants. Herein, we described a protocol for functional assessment of BRCA1 variants using CRISPR-mediated cytosine base editors that enable targeted C:G to T:A conversion in living cells.

使用 CRISPR 介质基础编辑器对 BRCA1 变体进行功能评估

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

医学

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

评估癌症药物过敏的有效性<em&gt;在体外</em&gt;和<em&gt;在体内</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

使用内源性野生型BRCA1基因的细胞表达，确定同源重组的BRCA1基因突变的影响

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

生物学

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

体外研究 用于靶向表观遗传沉默的工程化转录抑制因子的选择

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

生物工程

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 ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

基因组DNA富集转座为基础的技术的整个序列的农工商分析<em&gt; BRCA1基因</em&gt;<em&gt; BRCA2</em&gt;和9个基因参与DNA损伤修复

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

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评估癌症药物过敏的有效性

使用内源性野生型BRCA1基因的细胞表达，确定同源重组的BRCA1基因突变的影响

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择

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基因组DNA富集转座为基础的技术的整个序列的农工商分析

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沉默

摘要

探索更多视频

此视频中的章节

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

使用 CRISPR 介质基础编辑器对 BRCA1 变体进行功能评估

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

在乳腺癌细胞雌激素调节小分子RNA的剖析

评估癌症药物过敏的有效性

使用内源性野生型BRCA1基因的细胞表达，确定同源重组的BRCA1基因突变的影响

体外研究 用于靶向表观遗传沉默的工程化转录抑制因子的选择

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

基因组DNA富集转座为基础的技术的整个序列的农工商分析

免疫荧光DNA修复蛋白相互作用的可视化

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择