这项工作是由美国国立卫生研究院主任的新的创新奖计划（ED）的赠款支持。 AA是由美国国家癌症研究所和美国国家科学基金会本科奖项部分支持。

1.使病毒载体为感兴趣的基因，以及阳性和阴性对照的编码序列，插入pBABEpuro通过传统的克隆方法（PCR扩增，限制性消化和连接）。上有矢量4的限制性位点，其中该DNA可被插入:BamHI位，SnaBI位，EcoRI和SalI位。 <ol><li>从准备使用QIAGEN质粒Midi试剂盒感受态细胞转染级质粒DNA。 </li><li>使用NanoDrop2000分光光度计测量DNA浓度。 </li></ol> 2.逆转录病毒生产该开发平台-E包装细胞系将被用于产生亲嗜性逆转录病毒，将提供感兴趣的NIH 3T3细胞中的cDNA。 <ol><li>建立从冷冻细胞开发平台-E培养<ol><li>迅速解冻开发平台-E细胞在37℃水浴并转移到一个15毫升的锥形管。慢慢加入9毫升开发平台-E介质（杜lbecco改进的Eagle培养基（DMEM）+ 10％胎牛血清（FBS）+青霉素和链霉素）。 </li><li>离心管，在180× 克5分钟，并弃去上清液。 </li><li>重新悬浮细胞用10ml开发平台-E培养基并转移到10cm的培养皿中。 </li><li>孵育所述细胞在37℃，5％CO 2的培养箱中，直到它们80-90％汇合（约2天）。 </li></ol></li><li>细胞分裂<ol><li>吸出培养基，用PBS洗一次细胞。 </li><li>加2ml的0.05％胰蛋白酶-EDTA和孵化在室温1分钟。 </li><li>分离攻丝细胞通过手指，加入10 mL开发平台-E培养基中，将细胞悬液转移到15毫升管。 </li><li>离心管，在180× 克5分钟，并弃去上清液。 </li><li>悬浮细胞10毫升开发平台-E介质和种子在他们的新菜，在1:4-1:6稀释。 </li></ol></li><li>开发平台-E播种和转染<ol><li>种子2×10 6个细胞每使用开发平台-E中无任何抗生素10厘米培养皿。 </li><li>孵育在37°C，5％ 二氧化碳培养箱细胞O / N。 </li><li>次日，转移300微升的Opti-MEM的入1.5mL微管中。 </li><li>添加27微升的聚乙烯亚胺（PEI），具有成本效益的转染试剂12，向制备的管的Opti-MEM。通过手指敲击轻轻混匀，并培育5分钟在室温。 </li><li>添加9微克染同类的质粒DNA导入的Opti-MEM / PEI管，通过涡旋轻轻混匀，并孵育15分钟，在室温。 </li><li>添加DNA / PEI复杂滴进开发平台-E盘，孵化O / N在37℃，5％CO 2。 </li><li>第二天，吸出含有转染试剂的培养基中，并添加10ml新鲜开发平台-E培养基（无抗生素）。 </li><li>返回的细胞培养箱。 </li></ol></li></ol> 3. NIH 3T3细胞和病毒感染<ol><li>建立NIH3T3文化和播<ol><li>迅速解冻NIH 3T3细胞在37℃水浴中，并转移到15毫升管。慢慢加入9毫升NIH 3T3培养基（DMEM + 10％FBS）的。 </li><li>离心管，在180× 克5分钟，并弃去上清液。 </li><li>将细胞重悬于10毫升NIH 3T3介质和传输到10cm的培养皿中。 </li><li>孵育在37℃，5％CO 2培养箱中的细胞。这是非常重要的是，NIH 3T3细胞始终保持underconfluent（50-60％），作为自发变换（和灶形成的，因此基础水平）的频率增加，一旦培养达到汇合。 </li><li>每10 cm培养皿种子3×10 5细胞，并培育O / N感染的第二天。 </li></ol></li><li>感染<ol><li>通过使用10ml的一次性注射器，通过0.45μm孔径的尼龙膜过滤器过滤它，并将其转移到一个15毫升的收获从开发平台-E盘的逆转录病毒上清液管。 </li><li>加入10毫升新鲜开发平台-E中的细胞（无抗生素），并将其返回到孵化器。 </li><li>吸从NIH 3T3细胞中盘被感染的介质，并加入5ml含病毒过滤的上清液的定期NIH 3T3介质和5毫升。 </li><li>加入聚凝胺至培养皿以6微克/ ml的浓度。 </li><li>孵育细胞在o / n 37℃下，5％的CO 2。 </li><li>第二天，重复步骤3.2.1 - 3.2.5第二轮感染。 </li><li>更换含有病毒的培养基定期NIH 3T3中。 </li><li>允许在NIH 3T3细胞中生长2-3周，更换培养基是必要的。检查的目的蛋白质的表达通过常规蛋白电泳和免疫印迹上从复制平板全细胞裂解液。 </li></ol></li></ol> 4.染色和定量<ol><li>结晶紫染色<ol><li>吸出NIH 3T3中，将菜在冰上。 </li><li>用冰冷的PBS两次洗碗。 </li><li>固定的细胞用冰冷的甲醇中10分钟。 </li><li>从冰取出盘子，吸出甲醇，并加入3毫升0.5％结晶紫溶液中的​​25％甲醇（RT）下进行。 </li><li>孵育菜为5分钟在室温。 </li><li>吸出的结晶紫溶液并小心地冲洗用Milli-Q H 2 O的培养皿直至没有颜色在漂洗脱落。 </li><li>让菜干O / N在工作台上（发现）。 </li></ol></li><li>佛慈定量<ol><li>一旦菜是干的，用尺子来测量上盘细胞的深颜色的集合。只计数那些大于5毫米，直径为"焦点"。记录病灶的数量，并计算平均值和与对照相比的显着性。 </li></ol></li></ol>

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作者什么都没有透露。

在FFA提供了一种快速简便的方法来评估在体外恶性转化。它是适合于相对大数目的候选基因的筛选，并且其适度的技术要求，使其符合成本效益。此外，两个或多个基因可以共表达（有时被称为一个"合作"测定）来评价该组合的致瘤潜力。该测定的优势依赖于它的简单的技术，其易于定量和它的相对短的周转时间。然而，必须强调的是，它不构成所有的体外测定法的限制。该测定法?...

肿瘤细胞可以从他们的正常对应来区分由范围广泛的改变，从基因表达模式以表观基因组形态学和增生性改变。在后者，降低血清依赖性，接触（密度）抑制损失，收购的锚定非依赖性增殖，最终，当注射到动物形成肿瘤的能力是恶变3是有用的，可衡量的指标。几种体外和体内试验已经开发了用于细胞转化， 在体外测定的目的是识别测定改变培养形态（焦点形成实验），培养动力学（生长率，饱和密度）和生长因子（生长血清减少的）或锚地（生长在软琼脂）的要求。的黄金标准，用于确定细胞类型的恶性性质仍然肿瘤形成（异种移植物）中的实验动物。然而，T他花费和体内研究的长度并不总是使它们正当作为候选癌基因的第一个验证步骤或筛选。虽然没有在体外测定提供了一个基因的致癌潜力的一个明确的评估，它们提供的洞察致癌潜力，可能在体内研究缩小将来。其中最广泛使用的系统，用于在体外评估致癌潜力的是焦点形成分析2。这种方法依赖于使用的NIH 3T3小鼠成纤维细胞，非转化细胞系，显示强烈的接触抑制。在密度依赖性生长的丧失的癌基因的过度表达的结果;然后转化的细胞可以生长在多个层，形成"病灶"，很容易地可视化对未转化细胞的背景单层。焦点形成分析，那么，衡量一个候选人癌基因的能力引起恶变，就证明了接触INHIB损失银行足球比赛作为衡量的表型。 FFA的已使用蛋白激酶的过表达（ 例如 ，Src蛋白4，BRAF 5），转录因子（ 例如 ，N--myc基因6），G-蛋白偶联受体（ 例如 ，P2RY8 7）和GTP酶（ 例如 ，以评估转换，拉斯1），等等。相对容易此测定的使得它一个很好的选择，这将提供一个快速和视觉上明确的答案的基因的过表达是否足以转化的NIH 3T3小鼠成纤维细胞在体外 。 在这个协议中所描述的FFA使用开发平台-E包装细胞系8，它提供了病毒包装蛋白和逆转录病毒载体pBABEpuro 9（Addgene质粒1764）产生逆转录病毒。转染用含有感兴趣的基因的pBABEpuro构建体后，将开发平台-E的细胞系将产生亲嗜性逆转录病毒可以用于感染的NIH 3T3细胞。牛逼基因传递，他的病毒的方法比传统的化学转染方法更高效，它提供了一种可持续表达基因10。一旦掺入NIH 3T3细胞的基因组中，所关注的基因的过表达是由病毒长末端重复序列（LTR）启动子11驱动。这个常量表达式可以被用来确定感兴趣的基因是否具有致癌活性，如通过形成病灶，在NIH 3T3细胞测定。

<table><tbody><tr><td>pBABE-puro vector</td><td>Addgene</td><td>Plasmid 1764</td><td>cloning vector</td></tr><tr><td>Platinum-E Retroviral Packaging Cell Line, Ecotropic</td><td>Cell Biolabs, Inc.</td><td>RV-101</td><td>cell line for viral production</td></tr><tr><td>NIH 3T3 Cell Line murine</td><td>Sigma-Aldrich</td><td>93061524</td><td>cell line for focus formation assay</td></tr><tr><td>10 ml BD Luer-Lok tip syringe</td><td>BD Biosciences</td><td>309604</td><td>viral production reagent</td></tr><tr><td>0.45 &amp;mu;m Puradisc Syringe Filter</td><td>Whatman</td><td>6750-2504</td><td>viral production reagent</td></tr><tr><td>Polyethylenimine (PEI)</td><td>Polysciences, Inc.</td><td>23966-2</td><td>cell transfection reagent</td></tr><tr><td>Polybrene Infection / Transfection Reagent</td><td>EMD Millipore</td><td>TR-1003-G</td><td>cell transfection reagent</td></tr><tr><td>Crystal Violet</td><td>Fisher Scientific</td><td>C581-25</td><td>cell stain reagent</td></tr><tr><td>Plasmid Plus Midi Kit</td><td>QIAGEN</td><td>12945</td><td>plasmid purification</td></tr><tr><td>BD Falcon Tissue Culture Dishes</td><td>BD Biosciences</td><td>353003</td><td>cell culture supplies</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11995-065</td><td>cell culture media</td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td>cell culture supplies</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Gibco</td><td>31985-062</td><td>cell culture media</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>16000-044</td><td>cell culture media</td></tr></tbody></table>

focus formation: a cell-based assay to determine the oncogenic potential of a gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

MXD3是一个基本的螺旋 - 环 - 螺旋亮氨酸拉链（bHLHZ）转录因子是这样的MYC / MAX / MAD网络中的成员。它是MAD家族13-15的非典型构件，并且它已经被报道参与致癌16,17。相比pBABEpuro（阴性对照）和MYC（阳性对照），美国国立卫生研究院3T3菜肴其中MXD3过表达有显著更少灶（ 图1A）。在图1B中的数据是从多个实验收集的，以确定显着性。 有确?...

Watch this Scientific Journal Video about Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene at JoVE.com

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

聚焦形成分析提供了一个简单的方法来评估候选基因的转化潜力。

注重形成:一个基于Cell的检测，以确定一个基因的致癌性

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

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27.8K 次观看.  University of California, Davis. 聚焦形成分析提供了一个简单的方法来评估候选基因的转化潜力。 

视频: Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

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

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

集落形成细胞（CFC）的含量对人体的造血细胞

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

免疫与感染

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

c-fos 和 dusp1 在癌基因依赖性中的作用评价方法

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

酵母作为开发功能分析研究人类P53的机箱

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples from cancer patients often directly influences diagnosis, prognosis, and/or therapy selection. As a result, the accurate detection of gene fusions has become a critical component of clinical management for many disease types. Until recently, clinical gene fusion detection was predominantly accomplished through the use of single-gene assays. However, the ever-growing list of gene fusions with clinical significance has created a need for assessing fusion status of multiple genes simultaneously. Next generation sequencing (NGS)-based testing has met this demand through the ability to sequence nucleic acid in massively parallel fashion. Multiple NGS-based approaches that employ different strategies for gene target enrichment are now available for use in clinical molecular diagnostics, each with its own strengths and weaknesses. This article describes the use of anchored multiplex PCR (AMP)-based target enrichment and library preparation followed by NGS to assess for gene fusions in clinical solid tumor specimens. AMP is unique among amplicon-based enrichment approaches in that it identifies gene fusions regardless of the identity of the fusion partner. Detailed here are both the wet-bench and data analysis steps that ensure accurate gene fusion detection from clinical samples.

This article details the use of an anchored multiplex polymerase chain reaction-based library preparation kit followed by next-generation sequencing to assess for oncogenic gene fusions in clinical solid tumor samples. Both wet-bench and data analysis steps are described.

使用锚定多路聚合酶链反应的致癌基因融合检测,随后进行下一代测序

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

使用转录组学分析绘制无序致癌转录因子的结构-功能关系

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

医学

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

用于研究癌基因衍生的转录-复制冲突的邻位连接测定

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

Oncogene Expression Analysis with Alterations in pH in a Pancreatic Ductal Cell Line

The fourth leading cause of cancer-related death, pancreatic ductal adenocarcinoma (PDAC), has a 12% five-year survival rate. This disease has a poor prognosis and is characterized by a rigid stromal microenvironment, which represents a tangible challenge in its treatment. Chronic pancreatitis patients have a 10-fold greater risk of developing PDAC; in these patients, the ductal pH decreases from pH 8.0 to pH 6.0 due to bicarbonate insufficiency and the inflammatory milieu. Our goal was to understand the role of the acidic environment observed in chronic pancreatitis on oncogene expression in a pancreatic ductal cell line.
Therefore, 80% confluent human pancreatic ductal epithelial cells were incubated at pH 6.0 to pH 7.2 for 6 h. Total RNA from the cells was processed to enrich the total mRNA in the samples. Gene expression was evaluated via next-generation sequencing (NGS) of biological replicates. RNA-seq analysis was carried out with the aid of an online tool, and the differentially expressed genes (FCs &#60; &#177; 2.0) were identified; there were 90, 148, and 109 upregulated genes and 20, 14, and 23 downregulated genes at pH 6.0, 6.5, and 7.0, respectively. Four oncogenes were upregulated at pH 6.0, seven were upregulated at pH 6.5, and seven were upregulated at pH 7.0. The common genes that were upregulated at pH 6.0, pH 6.5, and pH 7.0 were lymphocyte cell-specific protein-tyrosine kinase (LCK) [pH 6.0, FC: 2.93; pH 6.5, FC: 2.93; pH 7.0, FC: 3.32], FGR proto-oncogene, Src family tyrosine kinase (FGR) [pH 6.0, FC: 4.17; pH 6.5, FC: 5.25; pH 7.0, FC: 5.09], and ArfGAP With SH3 domain, ankyrin repeat, and PH domain 3 (ASAP3) [pH 6.0, FC: 2.37; pH 6.5, FC: 3.84; pH 7.0, FC: 2.51]. The acidic environment triggers the activation of proto-oncogenes, which may trigger tumor initiation in chronic pancreatitis.

This study describes a protocol for exploring the effects of altered pH on oncogene expression via RNA-seq analysis of a pancreatic ductal cell line.

胰腺导管细胞系中 pH 值改变的癌基因表达分析

The fourth leading cause of cancer-related death, pancreatic ductal adenocarcinoma (PDAC), has a 12% five-year survival rate. This disease has a poor ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

软琼脂集落形成分析

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...