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 中稀释的腺相关病毒注射在 1 x 1012的。该病毒具有9型, 并包含在泛素助剂 (图 1) 下的Pten和蛋白表达的指南 RNA。Pten的丢失增加了前列腺上皮的 pAKT 和增生 (13)。</li>
	<li>在手术时使用雄性小鼠在8周的年龄, 以确保充分的前列腺发育。 注意: 本实验中显示的小鼠是在 C57BL/6J 背景12 (图 1) 上 Rosa26-LSL-Cas9-EGFP 敲老鼠。</li>
	<li>新鲜的准备一个一般的麻醉混合, 如果可能的话, 保持它不育。 注意: 为了更好的动物恢复手术后, 强烈建议麻醉解毒剂。<ol>
<li>要准备5毫升麻醉剂的总容量, 混合0.15 毫升 medetomidine 盐酸盐 (1 毫克/毫升), 0.4 毫升咪达唑仑 (5 毫克/毫升), 和0.25 毫升布托啡诺 (10 毫克/毫升) 与4.2 毫升无菌盐水水。 注: 替代麻醉方法, 如可吸入异氟醚, 可以根据机构动物护理指南使用。</li>
		<li>为了逆转盐酸 medetomidine 术后的效果, 将0.1 毫升 atipamezole (5 毫克/毫升) 与4.9 毫升无菌盐水混合使用。</li>
	</ol>
</li>
	<li>通过适当的消毒和灭菌, 准备无菌的外科手术环境。使用前用高压釜手术器械。</li>
</ol>2. 向小鼠前列腺提供病毒
<ol><li>麻醉用无菌1毫升注射器和 27G x ½针腹腔注射动物。使用麻醉剂量的0.01 毫升/克体重。如果手术持续时间超过30分钟, 则每30分钟注射1/3 的起始剂量即可维持麻醉。</li>
	<li>检查麻醉深度, 评估肌肉松弛, 踏板撤退, 和眼睑反射。当观察到反射丢失时, 剃掉动物的下腹部。</li>
	<li>用不育棉拭子小心地用兽医眼膏遮盖动物的眼睛, 以防止因角膜反射不足而失明。</li>
	<li>用70% 乙醇和10% 聚维酮碘擦拭剃腹, 对手术部位进行消毒。将手术区域从手术部位的中心擦洗到外围。</li>
	<li>使用无菌手术剪刀在低腹中线进行垂直皮肤切口, 约1厘米。</li>
	<li>用细点钳抬起腹膜, 以防止损坏下面铺设的器官。小心做一个 < 8 毫米切口使用手术剪刀通过腹膜。 注意: 与人类相比, 啮齿动物的前列腺包括四个单独的裂片。前叶附着于精囊。</li>
	<li>轻轻地将脂肪组织移开, 以发现精囊。使用环钳, 小心地抬起精囊, 直到前前列腺可以识别 (图 1)。</li>
	<li>使用0.5 毫升胰岛素注射器, 用 30G x 8 毫米针在前前列腺上皮细胞中注入30µL 病毒 (3 x 1010病毒微粒) 溶液。尽量减少渗漏, 确保液体在组织内吸收, 形成小气泡。把精囊放回腹腔。</li>
	<li>缝合腹膜与2-3 简单中断针使用6-0 可吸收缝线与13毫米, 3/8 圆, 和锥形点针。</li>
	<li>用3不育 4.8 x 6.5 毫米夹子将皮肤与镊子一起抬起, 以避免损伤腹膜。</li>
	<li>为了更好的恢复, 应用麻醉解毒剂在剂量0.01 毫升/克体重的腹腔注射使用无菌1毫升注射器和 27G x ½ "针。小心地把动物放回笼子里。</li>
</ol>3. 术后程序
<ol><li>在手术后, 保持笼子里的小鼠在加热垫上进行了1小时的治疗, 以防止体温过低直到完全恢复。如果使用解毒剂, 动物应在注射后几分钟内清醒。</li>
	<li>每天监视动物以得到适当的伤口愈合和疼痛征兆。如有必要, 根据机构动物护理指南, 管理额外的止痛药。</li>
	<li>一旦伤口闭合, 4-5 天后取下皮肤夹子。</li>
</ol>

<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. 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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. 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作者没有什么可透露的。

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)。由于病毒感染了几个单元格9, 此模型模拟了克隆扩展1415的人类方案, 并且是最佳的癌症研究 (图 2B)。
CRISPR/Cas9 技术的发现为体内基因编辑开辟了新的契机。现在有可能同时改变多个基因, 为这种异癌症提供了一种既节省成本又有时间效率的高价值技术。使用动物模型的研究得益于不仅在生殖, 而且在成人组织中产生基因改变的好处。此外, Cas9 表达小鼠的发展允许病毒传递的创可和 sgRNAs (图 1)。由于指南对基因组没有 Cas9 表达的影响, 这种病毒的工作是非危险的。
腺相关病毒诱发极低的免疫应答, 即使存在长达一年的时间, 病毒也不会集成到宿主基因组中, 这使得它更适合体内挖空研究16。在这方面, 必须指出, 不同的血清型可能会影响不同组织类型的转导效率。因此, 对靶向组织进行优化是实现高效率的关键。使用基因组集成慢病毒北疆可以另一方面允许长期癌基因表达10。
人前列腺不分离在不同的裂片, 并讨论了哪些小鼠前列腺叶最好代表人前列腺。当侧叶被提出时, 不同的裂片在肿瘤起动方面没有显著差异, 如9,17,18,19。病毒传递的另一个关键方面是体内其他细胞可能感染。我们观察到了转基因的前列腺基质细胞。在原位分娩过程中, 可以最大限度地减少风险, 避免血管的发生, 同时防止注射时出现渗漏。进一步的病毒设计, 包括前列腺特异启动子, 如 Probasin 启动子和血清型, 可能会增加细胞特异性。

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 (图 2B), 表示Pten13的丢失。pAKT 和 GFP 的免疫荧光联合染色识别了双阳性细胞 (图 2C)。这证实了腺相关病毒对前列腺细胞的转化。总的来说, 这些结果表明,在体内CRISPR/Cas9 基因编辑可以在前列腺上皮中使用腺相关病毒和 Rosa26-LSL-Cas9-EGFP 小鼠进行。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57525/57525fig1.jpg"> 图 1: 过程的说明.该过程是在普拉特et . 生成的 Rosa26-LSL-Cas9-EGFP 鼠标中进行的。12前前列腺 (AP) 附着于精囊 (SV)。病毒粒子表达引导 RNA 和一个创可蛋白被注入到前前列腺, 以改变基因表达。<a href="/files/ftp_upload/57525/57525fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57525/57525fig2.jpg"> 图 2: 通过原位病毒传递对小鼠前列腺进行基因改变.7周大的雄性 Rosa26-LSL-Cas9-EGFP 小鼠被注射与腺相关的病毒, 其中含有一个引导 RNA 的Pten和编码的一个创能蛋白。样本分析3月后, 病毒注射。(A) GFP 荧光成像的前前列腺。(B) 染色为 pAKT (褐色) 的组织学切片 (4 µm) 标记克隆区域, 并丢失Pten表达式。虚线框标志着高放大率的提交。(C) 荧光蛋白染色 (绿色) 和 pAKT (红色)。核酸被 DAPI 染色。左: 染色的未注射前叶显示没有信号的 GFP 或 pAKT。右: 染色的注射叶显示荧光蛋白 (细胞质染色) 和 pAKT (细胞膜定位) 的共同定位。(D) Ptenflox/flox小鼠注射了表达的腺病毒。在病毒传递6月后, 4 µm 前叶组织学切片的 H & E 染色。点状椭圆标志着高等级前列腺上皮内瘤样病变的面积。有关详细信息, 请参见引用9。<a href="/files/ftp_upload/57525/57525fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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。

CRISPR 指南对小鼠前列腺病毒传递的基因改变

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

virus-delivery-crispr-guides-to-murine-prostate-for-gene

Nanyang Technological University

Research

JoVE Journal

Cancer Research

8.6K 次观看.  Aarhus University. 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。

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

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

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. ...

科研

癌症研究

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis and to identify novel therapeutic targets. To investigate molecular events that drive the progression from primary cancer to metastasis, we have developed a bitransgenic mouse model, RIP-Tag; RIP-tva. In this mouse model, the rat insulin promoter (RIP) drives the expression of the SV40 T antigen (Tag) and the receptor for subgroup A avian leukosis virus (tva) in pancreatic &#946; cells. The mice develop pancreatic neuroendocrine tumors with 100% penetrance through well-defined stages that are similar to human tumorigenesis, with stages including hyperplasia, angiogenesis, adenoma, and invasive carcinoma. Because RIP-Tag; RIP-tva mice do not develop metastatic disease, genetic alterations that promote metastasis can be identified easily. Somatic gene transfer into tva-expressing, proliferating pancreatic &#946; premalignant lesions is achieved through intracardiac injection of avian retroviruses harboring the desired genetic alteration. A titer of &#62;1 x 108 infectious units per ml is considered appropriate for in vivo infection. In addition, avian retroviruses can infect cell lines derived from tumors in RIP-Tag; RIP-tva mice with high efficiency. The cell lines can also be used to characterize the metastatic factors. Here we demonstrate how to utilize this mouse model and cell lines to assess the functions of candidate genes in tumor metastasis.

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

We present a protocol to demonstrate a novel somatic gene transfer system utilizing RIP-Tag; RIP-tva mouse model to study the function of genes in metastasis. The avian retroviruses are delivered intracardiacally to ensure gene transfer into pre-malignant, noninvasive lesions of pancreatic &#946; cells in adult mice.

转移因子的鉴定和表征转移到新的RIP 标签;tva小鼠模型

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis ...

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer&#160;the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.

Resistance to cancer therapies contributes to disease progression and death. Determining the mechanistic underpinnings of resistance is crucial for improving therapeutic response. This manuscript details the protocol to generate taxane-resistant cell models of prostate cancer (PC) to help dissecting the pathways involved in progression to Docetaxel resistance in PC patients.

抗有丝分裂药物多西紫杉醇的前列腺癌细胞模型的建立

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

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

利用 CRISPR/Cas9 基因编辑研究细胞因子依赖性造血细胞中突变钙的致癌活性

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

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

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

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

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

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

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

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

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

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

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in 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, ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

骨肉瘤的三维骨细胞外基质模型

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

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

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

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 ...