Name: a double humanized blt-mice model featuring a stable human-like gut microbiome and human immune system
Uploaded: 2019-08-30T13:30:00
Description: Watch this Scientific Journal Video about A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System at JoVE.com

我们要感谢万燕民、康国斌和帕拉比·昆都协助生产BLThuman小鼠。我们要感谢UNMC基因组学核心基金，他们从内布拉斯加州功能基因组学研究网络获得部分支持，NE-INBRE P20GM103427-14，神经感官系统的分子生物学CoBRE P30GM110768，弗雷德和帕梅拉巴菲特癌症中心 - P30CA036727，根和红生物研究中心（CRRI） 36-5150-2085-20，内布拉斯加州研究计划。我们要感谢内布拉斯加大学林肯生命科学系及其工作人员的帮助。这项研究部分得到美国国家卫生研究院（NIH）资助R01AI124804、R21AI122377-01、P30 MH062261-16A1慢性HIV感染和衰老神经艾滋病（CHAIN）中心，1R01AI111862至Q Li的支持。 资助者在研究设计、数据收集和分析、编写手稿或决定出版方面没有作用。

此处描述的所有方法均根据内布拉斯加-林肯大学 （UNL） 批准的机构动物护理和研究委员会 （IACUC） 批准的协议进行。UNL的IACUC已经批准了两个与产生和使用hu-BLT小鼠有关的协议，包括双胡鼠。此外，UNL的科学研究监督委员会（SROC）还批准使用人类胚胎干细胞和胎儿组织，这些干细胞和胎儿组织是从高级生物科学资源采购用于人工化小鼠研究（SROC# 2016-1-002）。
1. 老鼠外壳和?...

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这里描述的方案是为创造双hu-BLT小鼠，具有功能性的人类免疫系统和稳定的人样肠道微生物群。该协议可以适应其他人性化或非人性化的小鼠模型，而无需GF动物和转基因设施。虽然此处描述的方法相对简单，但有几个关键细节对于成功创建双胡-BLT 小鼠非常重要。NSG小鼠具有极缺免疫性，预防感染是小鼠长期生存的关键。我们采取了以下措施来预防感染。首先，动物被安置在带有HEPA滤波器（0.22 ...

人化小鼠（胡鼠）已经改变了对人类健康和疾病的许多方面的研究，包括造脑、免疫、癌症、自身免疫性疾病和传染病1、2、3、4 ，5，6，7，8，9 。与其他小鼠模式不同，这些小鼠具有明显的优势，因为它们具有功能性的人类免疫系统，并且可能感染人类特定的病原体。然而，肠道微生物群的重要性已经通过它在许多人类疾病中的作用，如肥胖，代谢综合征，炎症性疾病和癌症10，11，12， 13.粘膜免疫系统和肠道微生物群受到相互调节，以保持肠道和系统平衡。免疫系统由肠道微生物群的抗原形成，免疫系统在促进共生肠道细菌和消除病原体方面起着重要的调节作用14、15然而，胡鼠的肠道微生物群没有很好地特征，鼠肠道微生物群在组成和功能上与人类有很大不同。这是由于鼠和人肠道之间的进化、生理和解剖学差异，以及饮食等其他重要因素，可能影响胡鼠病模型18的实验结果。因此，除了对胡鼠的鼠肠道微生物群进行分类外，还需要一种具有人类免疫系统和人类肠道微生物群的动物模型来研究人类疾病在体内的复杂相互作用。
直接在人类科目上研究人类疾病往往是不切实际的或不道德的。许多动物模型不能用于研究人类病原体，如人类免疫缺陷病毒1型（HIV-1）。非人类灵长类动物的遗传繁殖，非常昂贵，并且不易受许多人类病原体的影响。被衍生为无细菌（GF）并与类似人类的肠道微生物群重组的小鼠已被广泛用于研究人类健康和疾病。然而，这些动物没有人体免疫系统，与GF动物合作需要专门的设施、程序和专业知识。因此，需要改进临床前模型来研究肠道微生物群群与人体免疫系统的复杂关系。许多小鼠菌株，如NOD。Cg-PrkdcscidIl2rgtm1Wjl/SzJ （NSG），不作为 GF 商用。GF动物也可能遭受长期免疫缺陷，这是不能完全逆转的微生物的移植21。因此，我们创造了一种双胡鼠，具有功能性的人体免疫系统和稳定的人样肠道微生物群，在特定的无病原体（SPF）条件下。为了产生双胡鼠，对NSG小鼠进行了手术，以产生骨髓、肝脏、胸腺人化小鼠（hu-BLT）。然后用广谱抗生素治疗Hu-BLT小鼠，然后用健康的人类供体样本进行粪便移植。我们从45只双胡-BLT小鼠和4个人类粪便供体样本中抽取了173个粪便样本的细菌肠道微生物群。双胡-BLT小鼠具有独特的16S rRNA基因图谱，基于移植的个体人类供体样本。重要的是，移植的人类样微生物群在小鼠体内稳定，研究时间长达移植后14.5周。此外，预测的元基因组显示，双胡-BLT小鼠与更类似于人类供体样本的小鼠具有不同的预测功能能力。

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			<td height="21" style="height:21px;width:337px;">Animal Feeding Needles 18G</td>
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			<td style="width:103px;">9928B</td>
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			<td height="42" style="height:42px;width:337px;">Clidox-s Activator</td>
			<td style="width:225px;">Pharmacal Research Laboratories</td>
			<td style="width:103px;">95120F</td>
			<td></td>
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			<td height="42" style="height:42px;width:337px;">Clidox-s Base</td>
			<td style="width:225px;">Pharmacal Research Laboratories</td>
			<td style="width:103px;">96125F</td>
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			<td height="21" style="height:21px;width:337px;">DGM 108 cage rack</td>
			<td style="width:225px;">Techniplast</td>
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			<td height="42" style="height:42px;width:337px;">Flat Brown Grocery Bag 3-5/8&#34;D x 6&#34;W x 11-1/16&#34;L&#160;</td>
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			<td style="width:103px;">12R063</td>
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			<td style="width:225px;">OpenBiome</td>
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			<td height="21" style="height:21px;width:337px;">Grape Kool-Aid</td>
			<td style="width:225px;">Kraft Foods Inc.</td>
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			<td height="21" style="height:21px;width:337px;">hCD19-PE/Cy5</td>
			<td style="width:225px;">Biolegend</td>
			<td style="width:103px;">302209</td>
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			<td height="21" style="height:21px;width:337px;">hCD3-PE</td>
			<td style="width:225px;">Biolegend</td>
			<td style="width:103px;">300408</td>
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			<td height="21" style="height:21px;width:337px;">hCD4-Alexa 700</td>
			<td style="width:225px;">Biolegend</td>
			<td style="width:103px;">300526</td>
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			<td height="21" style="height:21px;width:337px;">hCD45-FITC</td>
			<td style="width:225px;">Biolegend</td>
			<td style="width:103px;">304006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">hCD8-APC/Cy7</td>
			<td style="width:225px;">Biolegend</td>
			<td style="width:103px;">301016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Lactate Buffered Ringer&#39;s Solution</td>
			<td style="width:225px;">Boston BioProducts Inc&#160;</td>
			<td style="width:103px;">PY-906-500&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">mCD45-APC</td>
			<td style="width:225px;">Biolegend</td>
			<td style="width:103px;">103111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Microvette 100 K3E</td>
			<td style="width:225px;">Microvette</td>
			<td style="width:103px;">20.1278.100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:337px;">Neosporin First Aid Antibiotic/Pain Relieving Ointment</td>
			<td style="width:225px;">Neosporin</td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ)</td>
			<td style="width:225px;">The Jackson Laboratory</td>
			<td style="width:103px;">005557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">PrecisionGlide 25 G Needle</td>
			<td style="width:225px;">BD</td>
			<td style="width:103px;">305127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">RS200 X-ray irradiator</td>
			<td style="width:225px;">RAD Source Technologies</td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Sealsafe Plus GM500 microisolator cages</td>
			<td style="width:225px;">Techniplast</td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Sterile Non-woven Gauze</td>
			<td style="width:225px;">Fisherbrand</td>
			<td style="width:103px;">22-028-558</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Teklad global 16% protein irradiated mouse chow</td>
			<td style="width:225px;">Teklad</td>
			<td style="width:103px;">2916</td>
			<td></td>
		</tr>
	</tbody>
</table>

a double humanized blt-mice model featuring a stable human-like gut microbiome and human immune system

具有功能性人体免疫系统的人类小鼠（胡鼠）从根本上改变了对人类病原体和疾病的研究。它们可用于模拟人类或其他动物模型中难以或不可能研究的疾病。肠道微生物群能对人类健康和疾病产生深远影响。然而，鼠肠道微生物群与人类发现的微生物群非常不同。 需要改进具有移植人类肠道微生物群的临床前小鼠模型。因此，我们创造了双胡鼠，具有人体免疫系统和稳定的人样肠道微生物群。点头。Cg-PrkdcscidIl2rgtm1Wjl/SzJ （NSG） 小鼠由于其免疫缺陷水平高，是人体化的最佳动物之一。然而，无细菌NSG小鼠和其他各种重要的无细菌小鼠模型目前尚未上市。此外，许多研究环境无法获得诺托生物设施，在诺生物条件下工作通常费用昂贵且耗时。重要的是，无细菌小鼠有一些免疫缺陷，即使在微生物移植后也存在。因此，我们开发了一种不需要无细菌动物或诺生物设施的协议。为了产生双胡鼠，NSG小鼠在手术前接受放射治疗，以产生骨髓、肝脏、胸腺-人化（hu-BLT）小鼠。然后，用广谱抗生素治疗小鼠，以耗尽先前存在的鼠肠微生物群。经过抗生素治疗，通过口服口腔进行粪便移植，并带有健康的人类供体样本。双胡-BLT小鼠根据移植的个体供体样本，具有独特的16S rRNA基因谱。重要的是，移植的人类样微生物群在移植后14.5周的研究期间在双胡-BLT小鼠中保持稳定。

图1显示了用于创建双胡-BLT小鼠的方法的大纲，并简要描述了在NSG小鼠中添加功能性人体免疫系统和稳定的人样肠道微生物群的过程。图2显示了术后10周对人化BLT-小鼠的外周血液进行流式细胞测定分析的例子。图3显示了用于转移肠道微生物群以创建双胡鼠的人类粪便供体样本的相对丰度。图 4显示了抗生素?...

Watch this Scientific Journal Video about A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System at JoVE.com

A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System

我们描述了一种产生双重人性化BLT小鼠的新方法，其特点是功能性人体免疫系统和稳定的人类样肠道微生物群。无需无细菌小鼠或诺生物菌设施即可遵循此协议。

具有稳定的人类状古特微生物群落和人类免疫系统的双人性化BLT小鼠模型

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be ...

我们的协议意义重大，因为它可用于复制产生具有人类肠道微生物群和人类免疫系统的小鼠。我们称这些老鼠为双人性化小鼠。双人性化小鼠模型可用于基础和转化生物医学研究，包括研究人类肠道微生物群与人体免疫系统之间的体内关系。演示该程序的将是博士后张建水和来自我们实验室的博士生张一伦。要将肝脏和胸腺组织植入左肾胶囊，首先将鼠标放入护罩中。在手术当天，以每克体重剂量12美分的剂量治疗全身亚致命照射小鼠。在确认适当水平的电前和中侧后，将鼠标在左侧和中侧周围剃须。将眼科软膏涂抹在动物的眼睛上，并根据需要涂抹耳标。用三种连续碘和70%异丙醇磨砂消毒皮肤。当皮肤已准备后，使用钳子将一至一至六毫米立方的人类胎儿肝脏组织片段加载到一个特罗卡中，然后将一到一至十六毫米的立方人类胎儿胸腺组织片段加载到三元体状体中。使用钳子抬起皮肤，并使用手术刀将小纵向切入皮肤。将鼠标左侧的切口扩展到 1.5 到 2 厘米。并使用钳子提升肌肉层。用剪刀纵向打开肌肉层，必要时延长切口，露出肾脏，轻轻抓住肾脏周围的脂肪组织。使用手术刀在肾胶囊的后端进行一到两毫米的切口，然后缓慢地将预装的小刀插入与肾脏长轴平行的切口中。释放肾胶囊和肾脏之间的组织，并返回肾脏和肠道到其正常位置。然后，使用缝合线关闭肌肉层和手术钉，以关闭皮肤。将鼠标放入自动杀菌的微隔离器保持架中，并监控直至完全保持。手术后6小时，将小鼠放在热灯下，用70%异丙醇对尾巴进行消毒。将200微升PBS中分离的1.5至5倍10至5个阳性的人类胎儿肝脏组织分离造血干细胞注射到每只动物一个扩张的尾静脉中。然后用温和的压力停止任何出血，将老鼠还回他们的家笼。手术后9至12周，使用适当大小的整形梳约束来隔离一只人性化小鼠的一条腿，并喷洒70%异丙醇的隔离腿的中侧。将抗生素和止痛软膏传播到收集现场。使用以 90 度角保持的 25 量针，刺穿沙芬静脉，将 50 到 100 微升血液收集到 EDTA 涂层的血收集管中。获得适当量血后，用无菌纱布向现场施加压力，以止血，然后再将鼠标返回到其笼中。然后使用采集的外周血样本，使用对人CD45、CD3、CD4、CD8、CD19和小鼠CD45的抗体，通过流式细胞测定来评估人体免疫细胞重组水平。对于新鲜的粪便样本收集，将个别的自 <3> <3>化纸袋放入烟罩中，将一只鼠标放入每个袋子中，直到每个动物排便。然后将小鼠返回到笼子，并使用无菌钳子将每个粪便样本转移到每只小鼠一个 1.5 毫升管中，以储存零下 80 摄氏度。在14天的抗生素治疗结束时，将饮用水更换为自动消毒水，将小鼠转移到新的自洗笼中。抗生素停止后24小时和48小时解冻适当准备的粪便微生物移植材料来源，并在厌氧条件下将样品在厌氧室中等。为了进行粪便移植，通过口服球菌将200微升的粪便微生物移植材料送到每个实验动物身上，并在人工小鼠的皮毛上或笼内床上用品上传播任何剩余的解冻物质。此处，显示了手术后 10 周人工 BLT 小鼠的外周血液流动细胞学分析示例。T和B细胞群都可以识别，也可以识别CD4和CD8阳性T细胞子集。在此图中，可以观察到用于转移肠道微生物群以创建双人性化小鼠的人类粪便供体样本的相对丰度。抗生素治疗引起的脾脏和塞库姆的表型变化与无细菌动物的表型变化相似。这一代表性的16S核糖体RNA测序数据的主要成分分析图显示，双人性化小鼠具有人类类的肠道微生物群系，这是人类供体样本所独有的。该协议最关键的方面是限制有害细菌的引入，以确保小鼠保持健康，并维持人类肠道微生物群。该模型具有推进个性化医学的潜力，并使我们能够研究人类疾病如何影响肠道微生物群，同时控制其他缓解因素。

Research

JoVE Journal

Biochemistry

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

一种用于在复杂的细胞群落稳定性同位素示踪排序亚群测量方法代谢

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

用于异构蛋白复合物 -  DNA相互作用研究的修饰的酵母一杂交系统

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

蛋白质消化、超滤和尺寸排除色谱法优选人血浆和血清外来体的分离

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

体外肠道宿主-微生物界面合成细菌联合的可行性评价

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

用于稳定碳氧同位素分析的牙釉质碳酸盐的取样与预处理

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

无目标液相色谱-质量光谱学-小麦粒基代谢代谢学分析

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Analysis and Specification of Starch Granule Size Distributions

淀粉颗粒大小分布的分析和规格

Starch from all plant sources are made up of granules in a range of sizes and shapes having different occurrence frequencies, i.e., exhibiting a size and ...