Name: defining gene functions in tumorigenesis by ex vivo ablation of floxed alleles in malignant peripheral nerve sheath tumor cells
Uploaded: 2021-08-25T14:00:00
Description: Watch this Scientific Journal Video about Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells at JoVE.com

这项工作得到了美国国家神经疾病和中风研究所（R01 NS048353，R01 NS109655），美国国家癌症研究所（R01 CA122804），国防部（X81XWH-09-1-0086，W81XWH-11-1-0498，W81XWH-12-1-0164，W81XWH-14-1-0073和W81XWH-15-1-0193）和儿童肿瘤基金会（2014-04-001和2015-05-007）的资助。

在与小鼠进行任何程序之前，所有程序必须由机构动物护理和使用委员会审查和批准。本手稿中描述的协议已获得南卡罗来纳医科大学机构动物护理和使用委员会的批准。该协议由经过适当培训的人员按照MUSC的机构动物护理指南执行。
1. 生成MPNSTs纯合子的 小鼠的Erbb4 flox 等位基因
<ol><li>产生 F1 代 P 0-GGFβ3;53+/-;埃尔布4f...

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这里介绍的详细方法是使用MPNST的GEM模型开发的。然而，如果感兴趣的肿瘤组织可以分散到单个细胞中，这些方法很容易适应GEM中出现的各种肿瘤类型。重要的是要确保絮状等位基因不会导致i）存活率下降，这可能使获得足够的小鼠难以筛查肿瘤，或ii）肿瘤潜伏期增加，可能使获得足够的荷瘤小鼠变得困难。如果絮状等位基因不影响存活，则两个队列的存活率将是相等的。如果它对肿瘤潜伏期?...

在过去二十年之前，人类癌症的治疗严重依赖放疗和化疗药物，这些药物通过破坏DNA或抑制DNA合成来广泛靶向快速增殖的细胞群。虽然这些方法确实抑制了癌细胞的生长，但它们对正常快速增殖的细胞类型（如肠上皮细胞和毛囊细胞）也有有害的副作用。最近，癌症治疗已经开始利用化疗药物，这些药物精确地靶向信号通路中的蛋白质，这对个体患者肿瘤的生长至关重要。这种方法通常被称为“精准医疗”，导致了不断扩大的单克隆抗体和小分子抑制剂库的发展。这些药物有效地抑制肿瘤细胞的增殖和存活，同时避免了常规化疗药物和放疗对正常细胞类型的有害副作用。用于治疗人类癌症的单克隆抗体最常靶向细胞表面分子，例如生长因子受体1 （例如，跨膜受体酪氨酸激酶的大家族）和免疫应答调节剂2 （例如，程序性细胞死亡蛋白1，程序性死亡配体1）。小分子抑制剂可以抑制细胞表面蛋白或位于细胞内的信号蛋白3。然而，为了有效地利用这些新的治疗剂，必须确定特定的癌症依赖于候选治疗剂所靶向的分子。
虽然这些新的治疗剂具有更集中的作用，但其中许多仍然抑制一种以上蛋白质的作用。此外，通常可以使用具有不同有效性和特异性的多种试剂来靶向特定蛋白质。因此，在临床前研究期间，明智的做法是使用其他方法（例如遗传消融）来验证候选蛋白质作为治疗靶标。验证蛋白质作为治疗靶点的一种特别有用的方法是在基因工程动物模型中消融编码候选蛋白质的基因，该模型开发特定的癌症类型。如果具有零突变（自然突变，基因工程零突变[“敲除”]或基因陷阱引入的零突变）的小鼠可用，并且小鼠存活到成年期，则这种方法可能相对简单。不幸的是，符合这些标准的具有零突变的小鼠通常不可用，通常是因为零突变导致胚胎死亡或在产后生命的最初几天死亡。在这种情况下，容易发生肿瘤型感兴趣的小鼠可以转而杂交到目的基因的关键片段被loxP位点（“floxed”）两侧的小鼠杂交，这允许通过将表达Cre重组酶的转基因引入肿瘤细胞（条件敲除）来消融该基因。此方法具有几个优点。首先，如果Cre驱动剂在肿瘤中表达，但不在导致常规敲除中死亡的细胞类型中表达，则这种方法可以潜在地验证候选治疗靶点。其次，在肿瘤细胞中烧蚀编码候选蛋白的基因，而不是在其他肿瘤内元素（如肿瘤相关成纤维细胞或免疫细胞）中，使研究者能够区分治疗靶点的细胞自主和非细胞自主效应。最后，他莫昔芬诱导的Cre驱动因子（CreERT2）允许研究者在肿瘤发展的不同阶段删除目的基因，并确定候选治疗剂最有可能有效的窗口。
不幸的是，还有一些技术问题可能会限制在GEM模型中出现的肿瘤中使用有条件敲除。例如，在肿瘤细胞中表达并避免生命所必需的正常细胞中基因缺失的Cre驱动器可能不可用。另一个可能被低估的问题是，Cre和CreERT2 驱动者经常可变地消融小鼠中絮状等位基因，导致GEM癌症中零突变的嵌合体。当这种情况发生时，靶向基因尚未消融的肿瘤细胞将继续快速增殖，用消融的等位基因过度生长肿瘤细胞。Cre驱动谱系中的马赛克化可能是由于目标谱系中非普遍的Cre表达以及独立于Cre表达4的单个细胞中的重组失败而发生的。这是Cre驱动因素的已知现象，与细胞类型有关，应在实验设计和数据解释期间考虑。Mosaicism可以掩盖敲除的影响，并导致研究人员错误地得出结论，即目的基因对肿瘤细胞增殖和/或存活不是必需的，因此不是有效的治疗靶点。
在之前的一项研究中遇到了其中一些问题，该研究试图确定受体酪氨酸激酶erbB4是否是MPNST细胞中的潜在治疗靶标5。在这些研究中，在施万细胞特异性髓鞘蛋白零启动子（P 0-GGFβ3小鼠）的控制下，使用表达编码神经肽-1（NRG1）同种型神经胶质生长因子-β3（GGFβ3）的转基因的小鼠。P 0-GGFβ3小鼠发展为多个丛状神经纤维瘤，通过概括常染色体显性肿瘤易感综合征1型（NF1）6型神经纤维瘤病的神经纤维瘤发病机制和丛状神经纤维瘤-MPNST进展的过程进展为MPNST。当与具有Trp53零突变的小鼠杂交时，所得P 0-GGFβ3;Trp53 +/-小鼠从头发展MPNSTs，如顺式Nf1 + / -;Trp53+/- 小鼠。
这些 MPNST 概括了在人类中从世界卫生组织 （WHO） II 级病变进展到 WHO IV 级病变7。在P 0-GGFβ3小鼠中，MPNSTs出现在三叉神经（58%）和脊髓背神经根（68%）7中预先存在的丛状神经纤维瘤内;在 P 0-GGFβ3 中产生的甲基物质免疫吸附剂;Trp53 +/-小鼠具有高度相似的分布。在人类中，MPNSTs最常出现在坐骨神经中，其次是臂丛神经，脊神经根，迷走神经，股骨，正中，骶丛，腘腘闭塞器以及胫骨后神经和尺神经8。这些GEM模型中的这种肿瘤分布与人类中的肿瘤分布有些不同。然而，在P 0-GGFβ3和P0-GGFβ3中出现的甲基卫星;Trp53 + / -小鼠在组织学上与人类MPNST相同，携带许多与人类MPNTS相同的突变，并概括了NF1患者中可见的神经纤维瘤 - MPNST进展过程。生成 P 0-GGFβ3 或 P0-GGFβ3;Erbb4-/-的Trp53+/-小鼠是不可行的，因为具有两个Erbb4空等位基因的小鼠在继发于心脏缺陷的胚胎第10.5天在子宫内死亡9。因为通过引入心脏特异性Erbb4转基因（α肌球蛋白重链（MHC）-Erbb4）来拯救心脏中的Erbb4表达导致活的Erbb4-/-小鼠10，产生具有复杂P 0-GGFβ3的小鼠;Trp53+/-α-麦格-埃尔布4;尝试了 Erbb4-/- 基因型。
然而，交配没有产生预期孟德尔比例的小鼠，表明所需的基因型是有害的。因此，生成P 0-GGFβ3;尝试使用具有絮状Erbb4等位基因11和CreERT2驱动程序的Trp53 + / -小鼠允许在这些小鼠中出现的MPNST中去除Erbb4。在这些动物中，许多具有完整Erbb4等位基因的肿瘤细胞仍然存在（嵌合体）。观察到的嵌合体可能是由于他莫昔芬递送效率低下造成的，这导致组织内重组效率的差异。自发代偿机制的可能性可以通过绕过Erbb4表达的要求，进一步促进他莫昔芬介导的重组中的嵌合体。Trp53的丢失使肿瘤细胞容易受到其他自发“允许”突变的影响，这可能会混淆对数据的解释。由于Erbb4完整的MPNST细胞似乎有可能掩盖在其他肿瘤细胞中烧蚀Erbb4的后果，因此这种方法被放弃了。
这些障碍导致了一种使用表达Cre重组酶和eGFP的腺病毒在非常早期的传代MPNST细胞中烧蚀Erbb4的方法。这些细胞可以使用FACS从未感染的细胞中分离出来，这显着减少了烧蚀的Erbb4基因的嵌合体。下面描述了用于实现这一目标的方法，以及用于评估基因消融在体外和体内效果的方法。以下方案是如何产生荷瘤小鼠的一个例子，这些小鼠在体内同种异体移植肿瘤生长评估之前产生携带目标胚胎致死基因的絮状等位基因的肿瘤。这包括描述用于分析Erbb4消融对体外肿瘤细胞增殖，存活和基因表达的影响以及原位同种异体移植物中的增殖，存活和血管生成的作用的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ad5CMV-eGFP</td>
			<td>Gene Transfer Vector Core, Univ of Iowa</td>
			<td>VVC-U of Iowa-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ad5CMVCre-eGFP</td>
			<td>Gene Transfer Vector Core, Univ of Iowa</td>
			<td>VVC-U of Iowa-1174</td>
			<td></td>
		</tr>
		<tr>
			<td>alexa 568 secondary antibody</td>
			<td>Thermo/Fisher</td>
			<td>GaR A11036</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioconductor Open Source Software for Bioninformatics</td>
			<td>Bioconductor</td>
			<td>http://www.bioconductor.org</td>
			<td>alternative statistical analysis tool used for step 4.4</td>
		</tr>
		<tr>
			<td>CD31</td>
			<td>Abcam</td>
			<td>ab28364</td>
			<td></td>
		</tr>
		<tr>
			<td>Celigo Image Cytometer</td>
			<td>Nexcelom Bioscience</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Stripper</td>
			<td>Corning</td>
			<td>25-056-Cl</td>
			<td>mixture of chelators</td>
		</tr>
		<tr>
			<td>DAB staining kit</td>
			<td>Vector Labs</td>
			<td>MP-7800</td>
			<td></td>
		</tr>
		<tr>
			<td>DAVID (Database for Annotation, Visualization, and Integrated Discovery)</td>
			<td>DAVID</td>
			<td>https://david.ncifcrf.gov</td>
			<td>functional enrichment analysis software used for step 4.5</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>15-013-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>DreamTaq and Buffer (Genotyping PCR)</td>
			<td>Thermo/Fisher</td>
			<td>EP0701 and K1072</td>
			<td></td>
		</tr>
		<tr>
			<td>erbB4 antibodies</td>
			<td>Santa Cruz</td>
			<td>sc-284</td>
			<td></td>
		</tr>
		<tr>
			<td>erbB4 antibodies</td>
			<td>Abcam</td>
			<td>ab35374</td>
			<td></td>
		</tr>
		<tr>
			<td>erbB4 antibodies</td>
			<td>Millipore</td>
			<td>HFR1: 05-1133</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Sorter</td>
			<td>BD Biosciences</td>
			<td>Aria II</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma</td>
			<td>F6886</td>
			<td></td>
		</tr>
		<tr>
			<td>GenomeSpace Tools and Data Sources</td>
			<td>GenomeSpace</td>
			<td>https://genomespace.org/support/tools/</td>
			<td>general resource for several types of open source bioinformatic tools for step 4.5</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Corning</td>
			<td>25-005-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Gorilla Gene Ontology enRIchment anaLysis and visuaLizAtion tool</td>
			<td>Gorilla</td>
			<td>N/A, http://cbl-gorilla.cs.technion.ac.il</td>
			<td>functional enrichment analysis software used for step 4.5</td>
		</tr>
		<tr>
			<td>GSEA Gene Set Enrichment Analysis</td>
			<td>Broad Institute</td>
			<td>N/A, https://www.gsea-msigdb.org/gsea/index.jsp</td>
			<td>functional enrichment analysis software used for step 4.5</td>
		</tr>
		<tr>
			<td>HSD: Athymic Nude-FOxn1nu mice</td>
			<td>Envigo (Previously Harlan Labs)</td>
			<td>69</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina HiSeq2500 (next generation DNA sequencer)</td>
			<td>Illumina</td>
			<td>Hi Seq 2500</td>
			<td>DNA sequencer used for step 4.2</td>
		</tr>
		<tr>
			<td>Lasergene: ArrayStar Gene expression and variant analysis</td>
			<td>DNAStar LaserGene software</td>
			<td>N/A</td>
			<td>software statistical and normalization analysis used for step 4.4</td>
		</tr>
		<tr>
			<td>Lasergene: SeqMan NGen sequence alignment assembly</td>
			<td>software alignment used for step 4.3</td>
			<td>N/A</td>
			<td>software alignment used for step 4.3</td>
		</tr>
		<tr>
			<td>Matrigel, low growth factor basement membrane matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>NRG1-beta</td>
			<td>In house</td>
			<td>Generated by SLC, also commercially available from R &#38; D Systems(396-HB-050/CF).</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear Stain Hoeschst 33342</td>
			<td>Thermo</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>Panther Gene Ontology Classification System</td>
			<td>Panther</td>
			<td>http://pantherdb.org</td>
			<td>functional enrichment analysis software used for step 4.5</td>
		</tr>
		<tr>
			<td>Partek (BWA aligner and analyzer)</td>
			<td>Partek, Ver 7</td>
			<td>N/A</td>
			<td>software alignment and statistical/normalization used for step 4.3</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer 1: 5&#8242;-CAAATGCTCTCTCTGTTCTTTGT 
			GTCTG- 3&#8242;</td>
			<td>Eurofins Genomics</td>
			<td>Primer 1 + 2: 250&#8201;bp ErbB4 null product and a 350&#8201;bp Floxed ErbB4 product;</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer 2: 5&#8242;-TTTTGCCAAGTTCTAATTCCATC 
			AGAAGC-3&#8242;</td>
			<td>Eurofins Genomics</td>
			<td>Primer 1 + 2: 250&#8201;bp ErbB4 null product and a 350&#8201;bp Floxed ErbB4 product;</td>
			<td></td>
		</tr>
		<tr>
			<td>
			Primer 3: 5&#8242;-TATTGTGTTCATCTATCATTGCA 
			ACCCAG-3&#8242;
			</td>
			<td>Eurofins Genomics</td>
			<td>Primer 1 + 3: 350&#8201;bp wild-type ErbB4 product.</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodine</td>
			<td>Fisher</td>
			<td>51-351-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteom Profiler Phospho-Kinase Arrays</td>
			<td>R&#38;D Systems</td>
			<td>ARY003B</td>
			<td></td>
		</tr>
		<tr>
			<td>Real time glo</td>
			<td>Promega</td>
			<td>G9712</td>
			<td>bioluminescent cell viability assay</td>
		</tr>
		<tr>
			<td>ToppGene Suite</td>
			<td>ToppGene</td>
			<td>https://toppgene.cchmc.org</td>
			<td>functional enrichment analysis software used for step 4.5</td>
		</tr>
		<tr>
			<td>Trizol (acid-quanidinium-phenol and choloroform based reagent)</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyramide Signal Amplification Kit</td>
			<td>Perkin Elmer</td>
			<td>NEL721001EA</td>
			<td></td>
		</tr>
	</tbody>
</table>

defining gene functions in tumorigenesis by ex vivo ablation of floxed alleles in malignant peripheral nerve sheath tumor cells

精确靶向人类癌症中关键蛋白质的新药的开发正在从根本上改变癌症治疗方法。然而，在使用这些药物之前，它们的靶蛋白必须被验证为特定癌症类型的治疗靶点。这种验证通常是通过在癌症的基因工程小鼠（GEM）模型中敲除编码候选治疗靶标的基因并确定其对肿瘤生长的影响来进行的。不幸的是，诸如传统淘汰赛中的胚胎杀伤力和有条件淘汰赛中的镶嵌性等技术问题经常限制这种方法。为了克服这些限制，开发了一种在GEM模型中产生的恶性周围神经鞘肿瘤（MPNSTs）短期培养物中烧蚀的未光斑胚胎致死性目的基因的方法。
本文描述了如何建立具有适当基因型的小鼠模型，从这些动物中提取短期肿瘤培养物，然后使用表达Cre重组酶和增强绿色荧光蛋白（eGFP）的腺病毒载体消融未发酵的胚胎致死基因。然后详细阐述使用荧光激活细胞分选（FACS）纯化用腺病毒转导的细胞，并定量基因消融对细胞增殖，活力，转录组和原位同种异体移植物生长的影响。这些方法为 在体外 和体内鉴定和验证治疗靶点提供了一种有效且可推广的方法 。 这些方法还提供了低传代肿瘤衍生细胞的可再生来源，减少了 体外 生长伪影。这使得靶向基因的生物学作用可以在各种生物过程中进行研究，例如由分泌组介导的迁移，入侵，转移和细胞间通信。

图4示出了转导P 0-GGFβ3时获得的典型结果;Trp53-/+;具有腺5CMV-eGFP腺病毒或AD5CMV-cre/eGFP腺病毒的Erbb4 fl/fl MPNST细胞（图4A）。用荧光显微镜观察培养物以鉴定表达eGFP的细胞，并通过相差显微镜确定同一场中存在的细胞总数为10x（顶部）和40x（底部）。如果需要，可以计算表达eGFP的细胞的百分比。代表性的FACS输出数据如...

Watch this Scientific Journal Video about Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells at JoVE.com

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

在这里，我们提出了一种方案，用于在基因工程小鼠模型衍生 的肿瘤中 执行胚胎在体内致命的基因敲除，然后评估敲除对肿瘤生长，增殖，存活，迁移，侵袭以及 体外 和 体内转录组的影响。

通过恶性周围神经鞘肿瘤细胞中絮状等位基因的 离体 消融来定义肿瘤发生的基因功能

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...

该协议提供了一种替代的体内方法来研究胚胎致死基因敲除的肿瘤生长作用。这种方法的优点是，通常胚胎致命的基因敲除可以在来自基因工程小鼠模型的肿瘤中进行，以研究它们在体内的功能丧失效应。该技术可应用于需要研究传统基因敲除小鼠模型中无法实现的胚胎必需基因的体内作用的疾病。生成动物模型需要时间，并且需要进行经验测试以确定腺病毒感染的最佳MOI，以确保实现适当的重组。首先，用所需的基因型识别小鼠。当荷瘤小鼠达到其人道终点时，对小鼠实施安乐死，并使用无菌手术刀切除肿瘤。使用手术刀将肿瘤解剖成面包式切口的三个部分，以产生用于福尔马林固定石蜡包埋的组织片段，早期传代培养生成和快速冷冻以进行分析分析。用苏木精和曙红对 5 微米厚的福尔马林固定、石蜡包埋的组织切片进行染色。一旦病理学家确认肿瘤，进行免疫组织化学染色以确认肿瘤诊断。将新鲜收集的恶性周围神经鞘肿瘤组织置于冰上冰冷的无菌PBS中，然后将其转移到无菌工作区以建立早期传代培养物。将肿瘤组织切碎成2至4毫米的碎片，并在含有10毫升生长培养基的10平方厘米处理的组织培养皿中研磨8至10次。将这些组织培养在高糖DMEM-10生长培养基中，其中补充有10个纳摩尔神经肽-1β和2个微摩尔毛喉素。将早期传代的肿瘤细胞以1.5倍的密度接种至第六细胞，每10平方厘米处理过含有DMEM-10生长培养基的10平方厘米的组织培养皿。12至16小时后，用2至4毫升DPBS洗涤贴壁培养物，并在10毫升无血清DMEM中以每细胞约400个斑块形成单位感染细胞。感染48小时后，在荧光显微镜上短暂检查细胞的EGFP信号，以确保有效感染约50%至100%阳性细胞，然后FACS对EGFP阳性感染细胞进行分类。FACS分选后，让细胞在组织培养箱中恢复至少24至48小时，然后准备细胞进行基于体外细胞的分析或体内移植。通过使用从部分分选的EGFP阳性细胞中分离的基因组DNA进行PCR来确认Erbb4缺失。在接下来的七天内，使用基于图像的自动细胞仪对接种在96孔板中的分选肿瘤细胞进行增殖测定。用钩子和碘化丙啶染料染色细胞，并使用自动读板器捕获细胞的图像，以计数每个孔中活细胞和死细胞的数量。使用标准酸性胍酚和氯仿方法从分选的肿瘤细胞中分离总RNA。打开软件，使用默认设置按照程序特定的步骤执行RNA序列比对。选择分析方法，寻找RNA，然后选择小鼠参考基因组。上传 BED 文件（如果排序内核提供）。上载 FASTQ 排序文件并为这些文件分配唯一的复制名称。组复制 FASTQ 文件，并将这些文件指定为复制集。为了进行统计和归一化分析，以识别具有强大统计能力的差异基因表达信号，打开分析软件并选择GFP FASTQ文件作为对照数据集。接下来，选择 DESeq2 作为统计和归一化方法，然后开始组装和分析。使用集成到开放获取功能富集分析工具中的基因本体数据集，对已鉴定的具有统计学意义的Erbb4介导的DEG进行功能富集分析，以确定Erbb4基因丢失的生物学和通路意义。将肿瘤细胞注射到麻醉小鼠的坐骨神经中，以评估感染后细胞中的体内同种异体移植物生长潜力。一旦肿瘤达到所需的大小，如前所述，将肿瘤从安乐死小鼠中解剖为两到三个部分。将一个组织切片固定在4%多聚甲醛中过夜，然后将固定切片包埋在石蜡中。从FFPE组织制作五微米厚的切片后，将切片安装在载玻片上，并使用H&E和免疫染色确认移植组织中是否存在肿瘤细胞，如前所述。为了确认两种实验条件之间的体内Erbb4表达差异，用Erbb4特异性抗体染色福尔马林固定的石蜡包埋组织。分析了恶性周围神经鞘肿瘤组织细胞与腺病毒的转导。使用荧光显微镜检测EGFP表达细胞，并使用相差显微镜测定细胞总数。在Cre介导的基因消融后，根据转染效率，与对照转导相比，PCR基因分型显示出完全的基因敲除和诱导和未引出的细胞的异质群体。在恶性周围神经鞘肿瘤组织的原位同种异体移植物中发现的特征性组织病理学与来自原始基因工程动物模型的肿瘤相比，显示出细胞形态的差异。Erbb4消融降低了腺5-CMV-克雷消融细胞中的细胞密度和细胞增殖，控制腺5-CMV-GFP转导细胞。与对照腺5-CMV-EGFP治疗的同种异体移植肿瘤相比，腺5-CMV-Cre转导的同种异体移植物的结果导致用腺5-CMV-Cre转导的肿瘤细胞中Erbb4的表达减少。通过对照腺5-CMV-Cre-转导细胞的免疫反应性检测评估血管密度，代表性结果显示，与对照腺5-CMV-EGFP转导细胞相比，腺5-CMV-Cre-转导细胞的同种异体移植物的血管密度显着降低。基因消融后，细胞可用于许多不同类型的基于细胞的测定，例如增殖，迁移，3D生长或微环境改变，以及体内原位注射。

Research

JoVE Journal

Cancer Research

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

该<em&gt;果蝇</em&gt;成虫盘瘤模型：基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

科研

癌症研究

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

神经入侵的体内小鼠坐骨神经模型

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

肿瘤靶向免疫调节副病毒的设计与评价

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

体内 通过脾T细胞流式细胞术筛选肿瘤来源的细胞外囊泡的免疫原性

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

离体 小鼠尿路上皮细胞腺病毒-Cre介导的基因缺失的类器官模型

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

来自切除人肿瘤标本的 离体 器官型腹膜假粘液瘤肿瘤切片的培养和成像

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of ...

Genome-Scale Screening to Identify and Compare Therapeutic Targets in Tumors

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Finding New Therapeutic Targets for Malignant Peripheral Nerve Sheath Tumor Through Genome-Scale shRNA Screens (Video) | JoVE

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

通过恶性周围神经鞘肿瘤细胞中絮状等位基因的 离体 消融来定义肿瘤发生的基因功能

通过恶性周围神经鞘肿瘤细胞中絮状等位基因的离体消融来定义肿瘤发生的基因功能