Name: genetic engineering of primary mouse intestinal organoids using magnetic nanoparticle transduction viral vectors for frozen sectioning
Uploaded: 2019-05-10T12:30:00
Description: Watch this Scientific Journal Video about Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning at JoVE.com

这项工作得到了国家卫生研究院(R01DK102943、R03CA182679、R03CA191621)、马里兰干细胞研究基金(2015-MSCRFE-1759,2017-MSCRFD-3934)、美国肺协会、Allegheny健康网络- 约翰斯的资助。霍普金斯研究基金和霍普金斯消化系统疾病基础研究核心中心。

该协议得到了约翰霍普金斯医疗机构动物护理和使用委员会(IACUC)的批准。该协议是从以前发布的方法10,11,12,13修改。
1. 试剂制备
<ol><li>提前几个小时准备新鲜的293T介质,在水浴中加热至37°C,使用前至少10分钟(表1)。</li>
	<li>准备质粒DNA2,13,14用于病毒包装。<sup class="xre...

<ol>
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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=TIGAR+is+required+for+efficient+intestinal+regeneration+and+tumorigenesis.">TIGAR is required for efficient intestinal regeneration and tumorigenesis.</a> Dev Cell. 25 (5), 463-477 (2013).</li><li>Xian, L., 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=HMGA1+amplifies+Wnt+signalling+and+expands+the+intestinal+stem+cell+compartment+and+Paneth+cell+niche.">HMGA1 amplifies Wnt signalling and expands the intestinal stem cell compartment and Paneth cell niche.</a> Nature Comm. , (2017).</li><li>Sato, T., 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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Koo, B. 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(120), (2017).</li><li>Mu&#241;oz, 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=The+Lgr5+intestinal+stem+cell+signature:+robust+expression+of+proposed+quiescent+'+4'+cellmarkers.">The Lgr5 intestinal stem cell signature: robust expression of proposed quiescent '+4' cellmarkers.</a> EMBO J. 31 (14), 3079-3091 (2012).</li><li>Schwank, G., 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=Functional+repair+of+CFTR+by+CRISPR/Cas9+in+intestinal+stem+cell+organoids+of+cystic+fibrosis+patients.">Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.</a> Cell Stem Cell. 13 (6), 653-658 (2014).</li><li>Andersson-Rolf, A., Fink, J., Mustata, R. C., Koo, B. 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成人肠道上皮作为有机物的初级培养物为研究干细胞功能、肠道上皮平衡和病理学1、2、3、病理的分子机制提供了强有力的工具。 4.虽然CRISPR/Cas9技术可用于基因工程有机物9,但它受到基于序列分析的广泛筛选和选择所需的广泛筛选和选择的限制。该协议的目的是提供清晰和简洁的说...

长时间将小鼠或人类墓穴细胞培养为小肠或结肠的三维 (3D) 有机体的能力提供了重大突破,因为这些培养物显示了肠道上皮在体内的决定性特征1,2,3.从原发墓穴衍生的有机体能够自我更新和自我组织,表现出与其原产组织相似的细胞功能。事实上,有机物不仅概括了体内墓穴的结构组织,而且概括了许多分子特征,为研究正常生物学和疾病状态提供了有用的工具。为了说明,器官研究揭示了参与组织再生1、2、3、4、5以及可增强功能的药物的新型分子途径。在病理设置6,7。
对肠道干细胞的研究特别感兴趣,因为肠道内衬是高度再生的哺乳动物组织之一,每3-5天更新一次,以保护生物体免受细菌、毒素和其他病原体的侵害。肠道流明。肠道干细胞(ISCs)负责这种非凡的再生能力,从而为研究成人干细胞功能1,2提供了独特的范例。在小鼠的沿线追踪实验表明,分离的Lgr5阳性干细胞可以培养出来,在体外产生3D器官或"迷你胃",在体外它们密切镜像体内的同类。有机体培养也可以从肠道密码细胞分离物中衍生,这些分离物由祖细胞、ISC 和 Paneth 细胞组成,后者构成体内的上皮利基细胞。事实上,来自原发性肠道密码细胞的有机体培养已经演变为一种相对常规的技术,在大多数实验室使用广泛可用的试剂很容易实现。该模型还适用于RNA测序(RNA-Seq)的基因表达定量分析,以及通过质谱法、免疫组织化学或免疫荧光染色2、4、8对蛋白质进行定量分析。此外,功能遗传学可以使用功能增益(基因过度表达或激活突变基因的表达)或功能丧失(基因沉默或功能丧失突变的表达)方法2。
重要的是,标准质粒DNA或多蛋白的病毒转导方案的低效率和高毒性仍然是该领域的一个主要障碍。虽然CRISPR/Cas9技术允许精确的基因组编辑,这种方法需要耗时的选择,然后是序列验证9。在这里,我们提出了一个病毒转导方案,用于初级肠道器官,通过结合到磁性纳米粒子和应用磁场来优化病毒颗粒的传递。对先前协议4、5、10、11、12、13作了关键修改,并提出提高效率的建议。我们还描述了一种从3D母体凝胶(今称为基底膜基质或基质)中培养的完整有机物生成冷冻部分的方法,以便通过免疫组织化学或免疫荧光染色进行进一步分析。

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td>Base medium for 293T cells</td>
		</tr>
		<tr>
			<td>DMEM/F12+</td>
			<td>Thermo Fisher Scientific</td>
			<td>12634010</td>
			<td>Base medium for organoid culture medium and organoid digestion buffer</td>
		</tr>
		<tr>
			<td>OPTI-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11058021</td>
			<td>Virus plasmids transfection medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35-011-CV</td>
			<td>Component of virus collection medium and 293T medium</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Component of organoid culture medium and crypt dissociation buffer</td>
		</tr>
		<tr>
			<td>PBS (without Ca2+, Mg2+)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010049</td>
			<td>A wash buffer and component of crypt dissociation buffer</td>
		</tr>
		<tr>
			<td>Mem-NEAA</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140050</td>
			<td>Component of organoid culture medium</td>
		</tr>
		<tr>
			<td>GlutamaxII</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>Component of organoid culture medium</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
			<td>Component of organoid culture medium</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Millipore Sigma</td>
			<td>E9644</td>
			<td>Component of organoid culture medium</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>Peprotech</td>
			<td>250-38 B</td>
			<td>Component of organoid culture medium</td>
		</tr>
		<tr>
			<td>R-spondin</td>
			<td>R&#38;D</td>
			<td>7150-RS-025/CF</td>
			<td>Component of organoid culture medium</td>
		</tr>
		<tr>
			<td>Human recombinant insulin</td>
			<td>Millipore Sigma</td>
			<td>I9278-5ml</td>
			<td>Component of organoid culture medium</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Millipore Sigma</td>
			<td>N3376-100G</td>
			<td>Component of Transduction medium</td>
		</tr>
		<tr>
			<td>Wnt3A</td>
			<td>R&#38;D</td>
			<td>5036-WN-010</td>
			<td>Component of Transduction medium</td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>Millipore Sigma</td>
			<td>Y0503-1MG</td>
			<td>Component of Transduction medium</td>
		</tr>
		<tr>
			<td>0.5M EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575020</td>
			<td>Component of Crypts dissociation buffer</td>
		</tr>
		<tr>
			<td>DTT (dithiothreitol)</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0861</td>
			<td>Component of Crypts dissociation buffer</td>
		</tr>
		<tr>
			<td>Dispase I</td>
			<td>Millipore Sigma</td>
			<td>D4818-2MG</td>
			<td>Component of organoid digestion buffer</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Millipore Sigma</td>
			<td>11284932001</td>
			<td>Component of organoid digestion buffer</td>
		</tr>
		<tr>
			<td>matrigel(Growth factor reduced)</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Used as a matrix to embed organoids</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td>Medium for transfection in viral production</td>
		</tr>
		<tr>
			<td>ViralMag R/L</td>
			<td>Oz Biosiences</td>
			<td>RL40200</td>
			<td>Magnetic particles of viral transduction</td>
		</tr>
		<tr>
			<td>Magnetic plate</td>
			<td>Oz Biosiences</td>
			<td>MF10000</td>
			<td>Magnetic plate to facilitate viral transduction</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher Scientific</td>
			<td>11668019</td>
			<td>Transfection agent in viral production</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Millipore Sigma</td>
			<td>A-003-E</td>
			<td>Coating for plates before seeding 293T cells</td>
		</tr>
		<tr>
			<td>4% Formaldehyde Solution</td>
			<td>Boster</td>
			<td>AR1068</td>
			<td>Solution to fix organoids</td>
		</tr>
		<tr>
			<td>O.C.T embedding compound</td>
			<td>Thermo Fisher Scientific</td>
			<td>4583S</td>
			<td>For embedding of the the organoids</td>
		</tr>
		<tr>
			<td>5 mL Falcon polystyrene tubes</td>
			<td>Corning</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon Tubes</td>
			<td>Sarstedt</td>
			<td>62.547.100</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbitron rotator II Rocker Shaker</td>
			<td>Boekel Scientific</td>
			<td>260250</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Inverted microscop CK30</td>
			<td>Olympus</td>
			<td>CK30</td>
			<td>for scanning and counting crypts</td>
		</tr>
		<tr>
			<td>Zeiss Axiovert 200 inverted fluorescence</td>
			<td>Nikon</td>
			<td>Axiovert 200</td>
			<td>for viewing fluorescence in the crypts</td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter unit with Ultracel-100 membrane</td>
			<td>Milipore Sigma</td>
			<td>UFC910024</td>
			<td>For concentrating viruses</td>
		</tr>
		<tr>
			<td>pluriStrainer 20 &#181;m (Cell Strainer)</td>
			<td>pluriSelect</td>
			<td>SKU 43-50020</td>
			<td>For preparing organoid fragments</td>
		</tr>
		<tr>
			<td>Falcon Cell Strainer</td>
			<td>Fisher Scientific</td>
			<td>352340</td>
			<td>For preparing cyrpts of similar size after crypt isolation</td>
		</tr>
		<tr>
			<td>Greiner CELLSTAR&#160;multiwell culture plates 48 wells (TC treated with lid)</td>
			<td>Millipore Sigma</td>
			<td>M8937-100EA</td>
			<td>ForD2:D37+D16:D37g organoid fragments</td>
		</tr>
		<tr>
			<td>Animal strain: C57BL/6J</td>
			<td>Jackson Lab</td>
			<td>#000664</td>
			<td>For organoid culture</td>
		</tr>
	</tbody>
</table>

genetic engineering of primary mouse intestinal organoids using magnetic nanoparticle transduction viral vectors for frozen sectioning

肠道器官培养为研究肠道干细胞和体外密码生物学提供了独特的机会,尽管操纵器官基因表达的有效方法在这一领域进展有限。虽然CRISPR/Cas9技术允许对细胞进行精确的基因组编辑,以便产生有机体,但这一策略需要通过序列分析进行广泛的选择和筛选,这既耗时又昂贵。在这里,我们提供了一个详细的方案,为有效的病毒转导肠道器官。这种方法快速、高效,从而减少了CRISPR/Cas9技术固有的时间和费用。我们还提出了一个协议,从完整的有机体培养物中生成冷冻部分,以便用免疫组织化学或免疫荧光染色进行进一步分析,可用于确认基因表达或沉默。在成功转导病毒载体进行基因表达或沉默后,可迅速评估肠道干细胞和密码功能。虽然大多数器官研究采用体外检测,但有机体也可以传送给小鼠进行体内功能分析。此外,我们的方法有利于预测对药物的治疗反应,因为目前可用的疗法通常通过调节基因表达或蛋白质功能而不是改变基因组来发挥作用。

在这里,我们描述了一种快速和高效的转导技术,该技术利用暴露于磁场的磁性纳米粒子,将慢病毒输送到感兴趣的细胞。利用现成的工具,我们不仅应用了这种方法来转导新鲜分离的密码细胞(图1A),还应用了有机物(图2)和其他耐火细胞,以用于更常规的转导方法。Lentiviral粒子可以很容易地与磁性纳米粒子结合,因此,通过使用磁板施加...

Watch this Scientific Journal Video about Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning at JoVE.com

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

使用磁纳米粒子转导病毒载体进行冷冻切片的原小鼠肠道器官的遗传工程

我们描述了分步说明:1) 使用磁性纳米粒子对慢透或抗逆转录病毒转导进行高效工程肠道有机体,以及 2) 从工程有机物生成冷冻部分。这种方法提供了一个强大的工具,有效地改变基因表达在有机体的研究下游效应。

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

由于器官可以重述原生结构组织，以及体内肠道上皮的许多分子特征，因此该技术使我们能够在实验室中研究正常的生物学和疾病状态。该技术的主要优点是能够使用非常高效的转导方法对器官进行基因工程，具有低细胞毒性，从而允许这些基因操纵的器官的功能研究。这种技术的影响延伸到肠道疾病的治疗，因为我们可以暴露肠道器官药物和其他药物或干预措施。此方法可用于研究其他系统中适合器官培养物的发育和病理过程，如乳房组织或在标准二维条件下生长的细胞。一般来说，在难以设计器官或其他细胞中，新进入这种方法的个体将成功地进行转导。当我们在转移植器官和干细胞时遇到困难时，我们首先想到了这种方法。这种方法的视觉演示至关重要，因为如果在嵌入过程中样品未得到适当处理，则可能会发生器官结构损失，以进行低温分段。为了培育用于病毒转导的器官，种子器官细胞培养物，每井在48井板中培养200微升转导介质，并在标准组织培养孵化器中孵育。解冻用于转导的病毒小瓶，允许约50微升的浓缩病毒，用于转导48孔板中每口井。在1.5毫升管中，将病毒与12微升的磁性纳米粒子溶液混合，在室温下孵育15分钟。15分钟后，将磁性纳米粒子溶液和病毒混合物加入要转导的细胞中。将48井板放在磁板上，并在标准组织培养培养箱中孵育至少两个小时。当病毒转染完成时，将受感染的器官细胞簇和转导培养素从每个井转移到1.5毫升管中。在500次g下离心5分钟。用温和的吸力丢弃上清液，冷却冰上含有颗粒的管子五分钟。在每个管中加入120微升的基底膜基质，然后缓慢上下移液，重新暂停颗粒。种子30微升滴基质细胞混合物到一个新的48井板。在37摄氏度下孵育板5至15分钟，直到基质凝固。在每个井中加入转导介质，并在标准组织培养培养箱中孵育三到四天。三到四天后，在10倍放大的光显微镜下检查培养物，以确保细胞簇组织成器官结构。用250微升的有机培养ENR介质轻轻更换每井中的转导介质。将盘子返回孵化器。通过轻轻吸气去除 ENR 介质开始此过程，小心不要扰动地下室膜基质。用 500 微升 PBS 轻轻清洗。通过每井添加一个4%的甲醛溶液的微升，并在室温下孵育30分钟，修复器官。30分钟后，通过吸气去除甲醛溶液。轻轻添加每口一微升的 PBS，然后轻轻吸气去除 PBS。以这种方式，用 PBS 洗两次。从第二次洗涤中去除 PBS，并在每个样品中加入一个 30% 蔗糖缓冲液的微升。在四摄氏度下在蔗糖中孵育固定有机物一小时，使样品脱水。通过吸气去除蔗糖缓冲液，并添加足够的嵌入化合物，以覆盖每个井中的基质层。在室温下孵育五分钟。接下来，将样品放在零下80摄氏度的冰柜中10分钟，或直到嵌入化合物变成固体和白色。将板与冷冻嵌入化合物在室温下放置，以便沿边缘将化合物熔化最小。使用手术刀或铲子将方块与井壁分开。快速工作以防止熔化，使用钳子去除基体嵌入复合块，并将其放在试样块中。用嵌入化合物完全填充模具。在零下 80 摄氏度下冻结 30 分钟，然后使用块进行剖分。该协议通过首先通过与GP相关的扁病毒载体的肠道器官进行转化进行了优化。GFP在器官发育的每个阶段都被可视化，包括早期当低温细胞组织成囊肿状结构时，或在后来的阶段，当器官形成芽。成功转导的肠道器官随后被正式固定，冷冻，并通过免疫荧光成像进行分析。在此图像中，核被染成蓝色，绿色表示上皮细胞粘附分子，从而划定细胞边界。红色表示利索索姆染色，以识别窗格细胞。一旦掌握，转染器官可以在大约三个小时内完成，器官的哭声嵌入可以在大约2个半小时内完成。尝试此过程时，在处理基底基质试剂时，必须记住在冰上工作。完成此过程后，可以进行免疫荧光分析和其他检测蛋白质或细胞器的方法，以评估定位或相对丰度。这项技术为研究人员探索体外的正常生物学和疾病状态铺平了道路，也为体内研究（如果转导的器官或细胞可以植入小鼠）铺平了道路。看完这段视频后，你应该对如何使用磁性标记的病毒进行基因工程，进行冷冻的器官。请不要忘记，使用病毒可能极其危险，执行此程序时应始终采取预防措施，例如佩戴适当的防护装备。

Research

JoVE Journal

Developmental Biology

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

使用非整合附加型质粒从冷冻血沉棕黄层诱导多能干细胞的产生

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

科研

发育生物学

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

从鼠耳和尾组织生成主成纤维细胞培养

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

心肌细胞DNA的合成和倍性的同时评价：一个方法来帮助心肌细胞再生和营业额的量化

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

Using Primary Neurosphere Cultures to Study Primary Cilia

使用主神经球文化研究纤毛

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

人肠道 Organoids 上皮屏障通透性的实时测量

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

小鼠肝外胆管有机体的产生

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

Generation of Multicellular Human Primary Endometrial Organoids

多细胞人类原发性子宫内膜器官的生成

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

使用纸屑小鼠研究矿化组织的克隆遗传追踪

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

使用胎儿肠道有机体在体外重述吸吮到发胶过渡

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...