Name: focus formation: a cell-based assay to determine the oncogenic potential of a gene
Uploaded: 2014-12-31T15:00:00
Description: Watch this Scientific Journal Video about Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene at JoVE.com

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<p class="jove...

<ol>
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P., Land, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+mammalian+gene+transfer:+high+titre+retroviral+vectors+with+multiple+drug+selection+markers+and+a+complementary+helper-free+packaging+cell+line.">Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line.</a> Nucleic acids research. 18, 3587-3596 (1990).</li><li>Kim, T. K., Eberwine, J. 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J., Wright, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-phase-specific+expression+of+the+Mad3+gene+in+proliferating+and+differentiating+cells.">S-phase-specific expression of the Mad3 gene in proliferating and differentiating cells.</a> The Biochemical journal. 359, 361-367 (2001).</li><li>Yun, J. S., Rust, J. M., Ishimaru, T., Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+of+the+Mad+family+member+Mad3+in+cerebellar+granule+neuron+precursor+proliferation.">A novel role of the Mad family member Mad3 in cerebellar granule neuron precursor proliferation.</a> Molecular and cellular biology. 27, 8178-8189 (2007).</li><li>Gore, Y., Lantner, F., Hart, G., Shachar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mad3+negatively+regulates+B+cell+differentiation+in+the+spleen+by+inducing+Id2+expression.">Mad3 negatively regulates B cell differentiation in the spleen by inducing Id2 expression.</a> Molecular biology of the cell. 21, 1864-1871 (2010).</li><li>Barisone, G. A., Yun, J. S., Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cerebellar+proliferation+to+tumorigenesis:+new+insights+into+the+role+of+Mad3.">From cerebellar proliferation to tumorigenesis: new insights into the role of Mad3.</a> Cell cycle. 7, 423-427 (2008).</li><li>Barisone, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+MXD3+in+proliferation+of+DAOY+human+medulloblastoma+cells.">Role of MXD3 in proliferation of DAOY human medulloblastoma cells.</a> PloS One. 7, e38508 (2012).</li><li>Nair, S. K., Burley, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+structures+of+Myc-Max+and+Mad-Max+recognizing+DNA.+Molecular+bases+of+regulation+by+proto-oncogenic+transcription+factors.">X-ray structures of Myc-Max and Mad-Max recognizing DNA. Molecular bases of regulation by proto-oncogenic transcription factors.</a> Cell. 112, 193-205 (2003).</li></ol>

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?...

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">pBABE-puro vector</td>
			<td style="width:127px;">Addgene</td>
			<td style="width:123px;">Plasmid 1764</td>
			<td style="width:171px;">cloning vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Platinum-E Retroviral Packaging Cell Line, Ecotropic</td>
			<td style="width:127px;">Cell Biolabs, Inc.</td>
			<td style="width:123px;">RV-101</td>
			<td>cell line for viral production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">NIH 3T3 Cell Line murine</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:123px;">93061524</td>
			<td>cell line for focus formation assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">10 mL BD Luer-Lok tip syringe</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:123px;">309604</td>
			<td>viral production reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">0.45 &#956;m Puradisc Syringe Filter</td>
			<td style="width:127px;">Whatman</td>
			<td style="width:123px;">6750-2504</td>
			<td>viral production reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Polyethylenimine (PEI)</td>
			<td style="width:127px;">Polysciences, Inc.</td>
			<td style="width:123px;">23966-2</td>
			<td>cell transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Polybrene Infection / Transfection Reagent</td>
			<td style="width:127px;">EMD Millipore</td>
			<td style="width:123px;">TR-1003-G</td>
			<td>cell transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Crystal Violet</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:123px;">C581-25</td>
			<td>cell stain reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Plasmid Plus Midi Kit</td>
			<td style="width:127px;">QIAGEN</td>
			<td style="width:123px;">12945</td>
			<td>plasmid purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">BD Falcon Tissue Culture Dishes</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:123px;">353003</td>
			<td>cell culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">11995-065</td>
			<td>cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">0.05% Trypsin-EDTA</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">25300-054</td>
			<td>cell culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">31985-062</td>
			<td>cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">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

注重形成：一个基于Cell的检测，以确定一个基因的致癌性

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。

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

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