Name: virus delivery of crispr guides to the murine prostate for gene alteration
Uploaded: 2018-04-27T15:30:00
Description: Watch this Scientific Journal Video about Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration at JoVE.com

MR 是由丹麦癌症协会 (R146-A9394-16-S2) 的一支奖学金资助的。MFB 和 MKT 由 AUFF 新星 (AUFF-E-2015-FLS-9-8) 资助。MR 和 MFB 由研究生院, 健康, 非盟共同出资。E.F.W. 实验室得到西班牙经济部 (SAF2015-70857, 由 ERDF-欧盟共同出资) 的赠款和一个紧急救济高级补助金 (741888-CSI 乐趣) 的支持。
我们要感谢莉莉法哈多梅勒 (基因、发育和疾病;国家癌症研究中心) 对手稿的批判性阅读。

该协议涉及实验室小鼠的手术方法。所有动物实验必须由一个机构动物护理和使用委员会 (IACUC) 单独审查和批准。由于该方法是基于动物的恢复和生存, 确保适当的麻醉, 疼痛管理, 并在任何时候无菌的外科环境。在手术期间, 使用加热垫防止体温过低, 直到麻醉恢复。
1. 开始考虑事项
<ol><li>根据使用的病毒, 不同的效价要求可能适用;相应地稀释病毒。 注: 在本例中, 在 PBS ?...

<ol>
	<li>Ferlay, J., 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=Cancer+incidence+and+mortality+worldwide:+sources,+methods+and+major+patterns+in+GLOBOCAN+2012.">Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012.</a> Int J Cancer. 136, E359-E386 (2015).</li><li>Gerlinger, M., 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=Intratumor+heterogeneity+and+branched+evolution+revealed+by+multiregion+sequencing.">Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.</a> N Engl J Med. 366, 883-892 (2012).</li><li>Robinson, D., 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=Integrative+Clinical+Genomics+of+Advanced+Prostate+Cancer.">Integrative Clinical Genomics of Advanced Prostate Cancer.</a> Cell. 162, 454 (2015).</li><li>Gama Sosa, M. A., De Gasperi, R., Elder, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+transgenesis:+an+overview.">Animal transgenesis: an overview.</a> Brain Struct Funct. 214, 91-109 (2010).</li><li>Wu, X., 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=Generation+of+a+prostate+epithelial+cell-specific+Cre+transgenic+mouse+model+for+tissue-specific+gene+ablation.">Generation of a prostate epithelial cell-specific Cre transgenic mouse model for tissue-specific gene ablation.</a> Mech Dev. 101, 61-69 (2001).</li><li>Ma, X., 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=Targeted+biallelic+inactivation+of+Pten+in+the+mouse+prostate+leads+to+prostate+cancer+accompanied+by+increased+epithelial+cell+proliferation+but+not+by+reduced+apoptosis.">Targeted biallelic inactivation of Pten in the mouse prostate leads to prostate cancer accompanied by increased epithelial cell proliferation but not by reduced apoptosis.</a> Cancer Res. 65, 5730-5739 (2005).</li><li>Chen, Z., 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=Crucial+role+of+p53-dependent+cellular+senescence+in+suppression+of+Pten-deficient+tumorigenesis.">Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis.</a> Nature. 436, 725-730 (2005).</li><li>Leow, C. C., Wang, X. D., Gao, W. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+method+of+generating+prostate-specific+Cre-LoxP+gene+switching+via+intraductal+delivery+of+adenovirus.">Novel method of generating prostate-specific Cre-LoxP gene switching via intraductal delivery of adenovirus.</a> Prostate. 65, 1-9 (2005).</li><li>Thomsen, M. K., 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=Loss+of+JUNB/AP-1+promotes+invasive+prostate+cancer.">Loss of JUNB/AP-1 promotes invasive prostate cancer.</a> Cell Death Differ. 22, 574-582 (2015).</li><li>Cho, H., 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=a+novel+GEM+model+for+metastatic+prostate+cancer+analysis+and+therapy,+reveals+myc+as+a+driver+of+Pten-mutant+metastasis.">a novel GEM model for metastatic prostate cancer analysis and therapy, reveals myc as a driver of Pten-mutant metastasis.</a> Cancer Discov. 4, 318-333 (2014).</li><li>Cho, H., 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=Rapid+in+vivo+validation+of+candidate+drivers+derived+from+the+PTEN-mutant+prostate+metastasis+genome.">Rapid in vivo validation of candidate drivers derived from the PTEN-mutant prostate metastasis genome.</a> Methods. 77-78, 197-204 (2015).</li><li>Platt, R. J., 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=CRISPR-Cas9+knockin+mice+for+genome+editing+and+cancer+modeling.">CRISPR-Cas9 knockin mice for genome editing and cancer modeling.</a> Cell. 159, 440-455 (2014).</li><li>Wang, S., 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=Prostate-specific+deletion+of+the+murine+Pten+tumor+suppressor+gene+leads+to+metastatic+prostate+cancer.">Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer.</a> Cancer Cell. 4, 209-221 (2003).</li><li>Liu, W., 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=Copy+number+analysis+indicates+monoclonal+origin+of+lethal+metastatic+prostate+cancer.">Copy number analysis indicates monoclonal origin of lethal metastatic prostate cancer.</a> Nat Med. 15, 559-565 (2009).</li><li>Haffner, M. C., 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=Tracking+the+clonal+origin+of+lethal+prostate+cancer.">Tracking the clonal origin of lethal prostate cancer.</a> J Clin Invest. 123, 4918-4922 (2013).</li><li>Daya, S., Berns, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+using+adeno-associated+virus+vectors.">Gene therapy using adeno-associated virus vectors.</a> Clin Microbiol Rev. 21, 583-593 (2008).</li><li>Price, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Aspects+of+Development+and+Structure+in+the+Prostate.">Comparative Aspects of Development and Structure in the Prostate.</a> Natl Cancer Inst Monogr. 12, 1-27 (1963).</li><li>McNeal, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+prostate:+an+historical+survey+of+divergent+views.">Anatomy of the prostate: an historical survey of divergent views.</a> Prostate. 1, 3-13 (1980).</li><li>Thomsen, M. K., 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=SOX9+elevation+in+the+prostate+promotes+proliferation+and+cooperates+with+PTEN+loss+to+drive+tumor+formation.">SOX9 elevation in the prostate promotes proliferation and cooperates with PTEN loss to drive tumor formation.</a> Cancer Res. 70, 979-987 (2010).</li></ol>

在本协议中, 我们描述了一种通过病毒注射改变小鼠前列腺前叶基因表达的方法, 为前列腺癌创建了一个强大的新的老鼠模型 (图 2)。在2005年的8中, 首次用利奥et描述了腺病毒的成功管理。我们以前已经展示了如何用腺病毒编码的 recombinase 蛋白可以取代耗时的杂交育种的一个培养的等位基因的组织特定删除9 (图 2D</stro...

在过去的十年中, 前列腺癌的检测和治疗有了显著的改善。然而, 随着预期寿命的延长, 前列腺癌的发病率也在增加。在全世界估计有110万例新病例, 这是男性癌症相关死亡的最常见原因之一1。前列腺癌的发展缓慢, 但当癌症进展到晚期转移状态时, 由于治疗选择有限, 预后较差。到目前为止, 只有少数基因被确定为常见的驱动程序在这个癌症, 其异质性和 multifocality 阻碍检测生物标志物和多弹头疾病驱动程序2,3。
传统的生成 GEMMs 的技术往往因其复杂性、及时的费用和成本而受到损害。条件敲除模型已被广泛用于研究前列腺癌候选基因, 这导致胚胎致死时, 灭活在生殖4。大多数常见的模型涉及前列腺特定的 recombinase 驱动由修改 Probasin5或 PSA6启动子集成在 GEMM 通过额外的杂交育种。在这些模型中, 感兴趣的基因将被瞄准大多数前列腺上皮细胞, 产生增生的整个器官, 这可能会损害动物的泌尿道功能7。
通过注射到小鼠前列腺前叶的病毒传递, 可以解决这个问题, 只针对几个细胞8。考虑到实验室的技术先决条件、专门知识和目标, 该方法从各种可能的变化中受益。成功的方法使用了针对 JunB和Pten9或慢病毒北疆目标的腺病毒的目标Pten和Trp53 10 已显示在其他人中。添加转基因, 如荧光素酶, 到病毒结构或 GEMM 将进一步通过生物发光成像的11, 使非侵入性监测疾病进展。
基于 CRISPR/Cas9 技术的基因组编辑揭示了通过快速生成体细胞挖空12来研究癌症的新的快速机会。单导 rna (sgRNAs) 定向到小鼠前列腺前叶的病毒传递建立了一个生理上更相关的前列腺癌模型。通过这种方法, 携带所选突变的单个细胞可以形成能够扩张和侵入的克隆。此外, 使用引导 rna 的多目标基因将产生细胞克隆与改变的不同基因。这将允许肿瘤的异质性和自然选择压力的癌症进展, 这可以揭示每个基因改变或上位机制的重要性。
在这里, 我们提出了一种方法, 提供病毒微粒的小鼠前列腺改变基因表达。小的腹部切口, 鼠前前列腺叶被暴露和病毒微粒被注射入裂片。手术后五天, 手术夹可从皮肤上取出, 前列腺癌可从8周后进行分析。总的来说, 这是一个快速和成本效益高的程序, 它对鼠标没有什么影响, 并允许较大的肿瘤发展, 而不损害鼠标。

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B6J.129(B6N)-Gt(ROSA)26Sortm1(CAG-cas9*,-EGFP)Fezh/J mice</td>
			<td>The Jackson Laboratory</td>
			<td>26175</td>
			<td></td>
		</tr>
		<tr>
			<td>0,5 ml U-100 insulin syringe</td>
			<td>BD</td>
			<td>324825</td>
			<td>for virus injection</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>BD</td>
			<td>300013</td>
			<td>for anesthesia injection</td>
		</tr>
		<tr>
			<td>30 G 1/2&#39;&#39; needle</td>
			<td>BD</td>
			<td>304000</td>
			<td>for anesthesia injection</td>
		</tr>
		<tr>
			<td>6-0 Polysorb Suture</td>
			<td>Medtronic</td>
			<td>GL889</td>
			<td>to close the peritoneum</td>
		</tr>
		<tr>
			<td>Disposable sterilized surgery drape</td>
			<td>multiple suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>multiple suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-Iodine Prep Pad</td>
			<td>Fisher Scientific</td>
			<td>06-669-70</td>
			<td></td>
		</tr>
		<tr>
			<td>Trimming machine</td>
			<td>Aesculap</td>
			<td>GT415</td>
			<td>to shave the abdomen</td>
		</tr>
		<tr>
			<td>Dumont Forceps</td>
			<td>F.S.T</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey Micro Needle Holder</td>
			<td>F.S.T</td>
			<td>12500-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>F.S.T</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow Pattern Forceps</td>
			<td>F.S.T</td>
			<td>11002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring Forceps</td>
			<td>F.S.T</td>
			<td>11106-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Wound Clip System Handle incl. Clips</td>
			<td>F.S.T</td>
			<td>12030-01</td>
			<td>to close the skin</td>
		</tr>
		<tr>
			<td>Wound Clip System Remover</td>
			<td>F.S.T</td>
			<td>12030-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td></td>
			<td>different models have been used</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Antisedan</td>
			<td></td>
			<td></td>
			<td>obtained from the animal facility</td>
		</tr>
		<tr>
			<td>Butorphanol</td>
			<td></td>
			<td></td>
			<td>obtained from the animal facility</td>
		</tr>
		<tr>
			<td>Medetomidinhydrochlorid</td>
			<td></td>
			<td></td>
			<td>obtained from the animal facility</td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td></td>
			<td></td>
			<td>obtained from the animal facility</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Fisher Scientific</td>
			<td>22-032-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>Takeda</td>
			<td>7242</td>
			<td></td>
		</tr>
		<tr>
			<td>Virus (AAV, AV)</td>
			<td>multiple suppliers</td>
			<td></td>
			<td>virus used in this protocol is an in-house production</td>
		</tr>
		<tr>
			<td>pAKT antibody</td>
			<td>CST</td>
			<td>4060</td>
			<td>used 1:200 dillution</td>
		</tr>
		<tr>
			<td>GFP anitbody</td>
			<td>CST</td>
			<td>2956</td>
			<td>used 1:100 dillution</td>
		</tr>
	</tbody>
</table>

virus delivery of crispr guides to the murine prostate for gene alteration

随着前列腺癌发病率的增加, 新的肿瘤驱动剂或调节剂的鉴定是至关重要的。基因工程鼠模型 (GEMM) 为前列腺癌的肿瘤异质性和其复杂的微进化动力学的阻碍。传统的前列腺癌小鼠模型包括生殖和条件性挖空、癌基因的基因表达和异种移植模型。在这些模型中生成 "从头" 突变是复杂、耗时且成本高昂的。此外, 大多数传统的模型都以前列腺上皮为目标, 而人前列腺癌则是众所周知的, 只在小的细胞子集中进化为孤立事件。有价值的模型不仅需要模拟前列腺癌的启动, 还要对晚期疾病进行进化。
在这里, 我们描述了一种方法来靶向几个细胞在前列腺上皮中的传感细胞的病毒粒子。对小鼠前列腺进行工程化病毒的传递, 可以改变前列腺上皮细胞中的基因表达。病毒类型和数量将据此定义基因改变的靶向细胞数量, 传感几个细胞用于癌症的启动和许多细胞进行基因治疗。通过手术为主的注射在前叶, 远端从泌尿道, 该模型的肿瘤可以扩大而不损害的泌尿功能的动物。此外, 通过仅针对前列腺上皮细胞的一个子集, 该技术能够使肿瘤的克隆扩张, 从而模仿人类肿瘤的启动, 进展, 以及通过基底膜的侵袭。
这项新技术提供了一个强大的前列腺癌模型, 改善生理相关性。动物的痛苦是有限的, 因为没有额外的育种是需要的, 整体动物计数减少。同时, 对新候选基因和通路的分析也加快了, 从而提高了成本效益。

为了评估病毒对小鼠前列腺的传递, 手术后三月分析了样本。Rosa26-LSL-Cas9-EGFP 小鼠12在暴露于病毒表达的培养蛋白的细胞中表达 GFP。用荧光显微镜检查前列腺样品, 以确定带有 GFP 信号的区域 (图 2A)。GFP 的信号表明前列腺上皮细胞的活性, 而不是 CRISPR 引导的基因编辑是否被诱导。免疫组化部分显示高表达 pAKT 的细胞的焦点区域 (<stron...

Watch this Scientific Journal Video about Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration at JoVE.com

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

该协议描述了一种新建立的方法, 病毒传递到小鼠前列腺。使用 CRISPR/Cas9 技术, 基因过度表达, 或 recombinase 分娩, 该技术允许基因表达的原位改变, 并实施了新的小鼠模型前列腺癌。

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疲劳的小鼠模型的外围照射引起

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

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Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

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Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

舌细胞外基质的制备与舌鳞癌体外重建

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

雄激素依赖性和去势抗性前列腺癌的荧光自体小鼠模型

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

磷酸浓缩结合无标签定量质谱法研究前列腺癌的 Phosphoproteome

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Preparation of Exosomes for siRNA Delivery to Cancer Cells

用于向癌细胞输送 sirna 的外质体的制备

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

利用有机体培养评价小鼠前列腺皮细胞的分化能力

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...