在Live主机转利什曼原虫寄生虫在体内成像
1Interdisciplinary Immunology Program,University of Iowa, and the VA Medical Center, 2Department of Biochemistry,University of Iowa, and the VA Medical Center, 3Department of Internal Medicine,University of Iowa, 4Department of Molecular Microbiology,Washington University School of Medicine, 5Division of Dermatology,Harbor-UCLA Medical Center, Hanley-Hardison Research Center, 6Interdisciplinary Immunology Program,Iowa City VA Medical Center, 7Departments of Internal Medicine, Microbiology and Epidemiology,University of Iowa
Summary
一个<em>在体内</em>成像系统是用来生成的定量测量与Trypanosomatid原虫感染小鼠<em>利什曼原虫</em>。这是一个慢性的过程中整个检测在许多组织中表达的荧光素酶的寄生虫的非侵入性和非致命性的方法<em>利什曼原虫</em> SPP。感染。
Abstract
独特品种<em>利什曼原虫，</em>一个家庭的原生动物寄生虫<em> Trypanosomatidae</em>，通常会导致不同的人类疾病的表现。疾病最常见的形式是内脏利什曼病（VL）和皮肤利什曼病（CL）。利什曼病的小鼠模型被广泛使用，但在小鼠疾病的寄生虫负担的量化要求的小鼠在感染后的不同时间安乐死。寄生负载，然后在显微镜下测量，限制稀释实验中，或寄生虫的DNA定量PCR扩增。 “<em>在体内</em>成像系统（IVIS）有一个集成的软件包，使细胞在生物体相关的发光信号的检测。既可以减少动物的使用和纵向遵循感染在体内成像模型在个人，<em>利什曼原虫</em> SPP。造成VL或CL成立。寄生虫设计，以表达荧光素酶，这些被引入到小鼠皮下或静脉注射。转基因荧光素酶驾驶的生物发光的定量测量<em>利什曼原虫</em使用IVIS>鼠标内的寄生虫。个人小鼠可成像多次在纵向研究中，让我们评估在最初的实验寄生虫接种的动物间的变化，并评估在小鼠组织中的寄生虫的繁殖。寄生虫检测灵敏度高，与皮肤的位置。虽然这是极有可能的信号（光子/秒/寄生虫）是在更深的内脏器官比皮肤低，但信号的定量比较肤浅与深刻的网站还没有完成。这是可能的部位的寄生虫之间的数字不能直接比较，但可以在同一组织中的寄生虫负载之间的小鼠相比。一个visceralizing物种的例子（<em> L。 infantum chagasi</em>）和一个物种造成皮肤利什曼病（<em> L。墨西哥</em>）所示。 IVIS程序可用于监测和分析各种各样的小动物模型<em>利什曼原虫</em>物种造成人类利什曼病的不同形式。
Protocol
1。小动物转基因利什曼原虫感染 1。寄生虫线转基因利什曼原虫 SPP。寄生虫表达荧光素酶的生成使用episomal或作为一个整合载体。1 2克隆线是首选。两个要点是： （一）综合超过episomal荧光素酶的荧光素酶是首选，因为这些寄生虫线，在理论上应该更好地保留在药物的压力，即没有引入到哺乳动物的基因。然而，?…
Discussion
在活体成像系统（IVIS），为整个动物的成像或体内成像实验感染利什曼病的不同形式的模型的 方法。18,16利什曼原虫SPP。寄生虫，可设计，以表达萤火虫荧光素酶在体内 IVIS成像技术检测的水平。这种方法的主要优势之一是它允许利什曼原虫属非侵入性可视化。活的动物宿主内。该方法已应用于其他传染性疾病模型，传染性病原体可以表达的转基因?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是从退伍军人事务部的优异审查授予部分经费，由美国国立卫生研究院拨款AI045540，AI067874，AI076233 – 01和AI080801（水电部），和AI29646（SMB）。这项工作是在CT和JG的经费由美国国立卫生研究院的T32 AI07511部分。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| D-Luciferin Potassium Salt | Reagent | Caliper LifeSciences (Formerly Xenogen) | 122796 | |
| IVIS Imaging System 200 Series | Equipment | Caliper LifeSciences | Other IVIS models that can be used include: Lumina II, Lumina XR, Kinetic, and Spectrum. |
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Tags
In Vivo ImagingTransgenic Leishmania ParasitesLive HostLeishmania SpeciesVisceral LeishmaniasisCutaneous LeishmaniasisMouse ModelsParasite BurdensMicroscopyLimiting Dilution AssayQPCR AmplificationIn Vivo Imaging System (IVIS)Bioluminescent SignalLuciferase ExpressionIntradermal InjectionIntravenous InjectionLongitudinal StudiesInter-animal VariationParasite InoculaMultiplication Of ParasitesHigh SensitivityCutaneous Locations
Citar este artigo
Thalhofer, C. J., Graff, J. W., Love-Homan, L., Hickerson, S. M., Craft, N., Beverley, S. M., Wilson, M. E. In vivo Imaging of Transgenic Leishmania Parasites in a Live Host. J. Vis. Exp. (41), e1980, doi:10.3791/1980 (2010).