Summary

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

Published: January 23, 2014
doi:

Summary

我们原位杂交协议建立一个荧光供于动物模型的组织切片中的持久DNA病毒基因组的检测。这个协议允许研究感染过程由codetection病毒基因组,它的RNA产物,以及单细胞内的病毒或细胞蛋白。

Abstract

的基因的单细胞codetection,其RNA产物和细胞调节蛋白是关键的,研究基因表达的调控。这是在病毒学领域的挑战,特别对于核复制的持久性DNA病毒,涉及动物模型的研究。单纯疱疹病毒1型(HSV-1)建立在神经元末梢终身潜伏感染。潜隐病毒作为水库,从它重新激活并诱导新的疱疹发作。的HSV-1的等待时间的细胞生物学仍知之甚少,部分原因是由于缺乏方法来检测HSV-1基因组原位的动物模型。我们描述了DNA的荧光原位杂交(FISH)方法有效地检测来自被感染的动物模型神经组织切片,在低拷贝病毒的基因组。该方法依赖于基于热的抗原修复,和直接标记的自制的DNA探针,或可商购的探针。我们开发了一个三重的领带宁方法,结合DNA-FISH与RNA-FISH和免疫荧光,使用基于过氧化物酶信号放大,以适应每个染色要求。一个主要的改进是获得,在10μm的组织切片,低背景信号,可以在高分辨率的共聚焦显微镜和宽视场常规落射荧光成像的能力。此外,三重染色加工具有广泛的针对细胞和病毒蛋白质的抗体。完整的协议需要2.5天,适应组织内抗体和探针的渗透。

Introduction

单纯疱疹病毒1型(HSV-1)是一种持久性人神经病毒,在周围神经系统,从它周期性地重新激活的复制和蔓延的三叉神经节(TG)的神经元建立长期潜伏感染。在HSV-1基因组是一个150 kb的双链DNA本地化主机神经元那里仍然是多拷贝chromatinized质粒,不整合到宿主细胞基因组1,2的核心。期间延时,HSV-1复制周期的遗传程序,强烈抑制,基因表达被限制在潜伏相关转录物(LAT)基因,从延迟建立到活化3的起始。土地增值税产生加工成一个主要的2 kb的稳定套索长8.5 kb的非编码RNA,和几个miRNA的4-7。因此,HSV-1的延迟为特征的病毒基因组DNA,RNA LAT,以及没有可检测的复制周期蛋白的存在。

ontent“>动物模型,主要是鼠和兔,是实验模型扼要的人为延迟的几个特点,其中之一这些模型的主要利益是他们让学习HSV-1潜伏在免疫活性宿主的生理方面的问题。在过去的几十年许多实验性的工具,如遗传修饰的病毒和小鼠中,已经开发研究生理学,遗传学和HSV-1的延迟,从动物组织中的细胞生物学。到现在为止,病毒基因组DNA的检测和定量通过Southern印迹和从定量PCR分离甘油三酯,然而,目前还没有检测HSV-1基因组原位杂交组织切片8,因此,延迟是经常通过土地增值税的RNA的检测采用原位杂交的RNA,而不是评估的组织切片方法可用病毒基因组的检测,因为它已经不可能表征感染细胞中基于病毒的基因组的存在下,噻的技术限制是一个主要的缺点的宿主-病毒相互作用,例如病毒基因组和细胞和病毒基因的表达,或在宿主细胞介导的免疫应答9,10之间的关系的许多方面进行分析。

最重要的是,在潜伏感染的细胞-细胞间的异质性保持相对未开发的,并已被证明是延迟的一个重要特征在小鼠和在植入到SCID小鼠中11-17人体感觉神经节的神经元。通常情况下,它表明通过qPCR即每个细胞的HSV-1的基因组拷贝数变化从5到几百。尽管土地增值税表现为潜伏期和活化的关键调节因子,对离体神经元和原位 PCR定量PCR的数据显示,潜伏感染神经细胞的一个子集,低至30%,体现了土地增值税轨迹11,12,18-21。如何在宿主细胞内,并在病毒潜伏期成立组织影响的细胞环境和病毒基因表达仍不清楚。这里我们描述了原位杂交(FISH)方法用于有效地检测的动物的神经组织切片内的低拷贝的HSV-1基因组DNA的荧光健壮。这种方法已被设计和使用我们进入到高分辨率显微成像也就是要研究的病毒基因组的相互作用与宿主细胞内的核部件22。此外,我们描述了用于同时检测病毒DNA与RNA和蛋白质,这是一个独特的工具来描述,调节病毒基因表达的病毒 – 宿主相互作用的倍数染色方法。该方法也可以应用于范围广泛的要求的检测HSV-1的潜伏基因组中,如在大量切片的量化被感染的神经元的分析。一个关键的步骤是应用抗原修复治疗,使病毒DNA访问杂交。因此,该协议也可能是有效的,以检测其他双链DNA病毒,这是目前无法检测动物组织中传统的DNA-FISH方法。

Protocol

这种方法被用于先前22发表的一项研究。对于一般的背景和常规操作的描述在ISH,IF和鱼,我们建议如下现有的文献23。 1。动物感染所有涉及实验动物的程序符合伦理问题的研究协会在视觉与眼科在研究中使用的动物(ARVO)声明,并批准了UPR-3296-CNRS的地方伦理委员会,按照欧洲共同体理事会指令86/609/EEC。所有动物无限制的获得食物和水。…

Representative Results

经过几个月的广泛测试,我们发现,热基化学揭露原位杂交技术制成潜伏的HSV-1基因组可用于荧光。在这个过程中,我们尝试了各种去掩蔽过程,并仅基于热的处理( 即加热部分到子沸点温度在微波炉中)出现的效率。然后,我们测试所使用的常规免疫组织化学(IHC)和电子显微镜来检索表位31,32,包括0.01M的pH值为6.0柠檬酸缓冲液(我们在所有的研究中使用),1×PBS中,1mM?…

Discussion

这里所描述的协议允许的小鼠神经组织切片的神经元内HSV-1潜伏基因组的检测。我们的调节病毒基因表达的途径的理解已经由缺乏方法来检测HSV-1基因组DNA 的原位神经组织内的限制。基因组拷贝数和被感染的神经元比例的信息主要来自PCR分析对分离的神经元11,12。在阐明作用的宿主细胞的核架构对HSV-1的延迟,我们设置确定潜伏的HSV-1基因组的国产化通过DNA-FISH,潜伏感染的小鼠神经?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢N.萨维特尔(辛辛那提儿童医院医学中心,辛辛那提,俄亥俄州,美国),S. Efstathiou(英国剑桥的大学)和詹姆斯·希尔(路易斯安那州立大学健康科学中心,新奥尔良,美国)提供的样品从HSV-1感染的小鼠和兔子分别和试剂; H.增本(上总DNA研究所,千叶县,日本)和S. Khochbin(阿尔贝研究所Bonniot,格勒诺布尔,法国)的有益讨论。

这项工作是由该中心法国国家科学研究(CNRS)(ATIP程序,PL,http://www.cnrs.fr)资助,法国国家研究署(ANR)(ANR-05-MIIM- 008-01,CENTROLAT, http://www.agencenationale-recherche.fr ),该FINOVI基金会( http://www.finovi.org/:fr:start ),该LabEX DEVweCAN(ANR-10的LabX-61大学德里昂),在程序中“InvestissemenTS D'艾文莉“(ANR-11-IDEX-0007)的ANR操作( http://www.agence-nationale-recherche.fr ),L'协会倒拉RECHERCHE CONTRE乐巨蟹座(ARC-7979和ARC- 4910, http://www.arc-cancer.net ),LA法甲驳国立癌症勒(LNCC, http://www.ligue-cancer.net ),和印加(EPIPRO程序, http://www.e – cancer.fr ),FC和PL是法国国家科学研究中心的研究人员。

Materials

Balb/c mice Janvier, France 6 week-old females
HSV-1 strains SC16 strain (wild type) See Labetoule, M. et al. (2003) Invest Ophthalmol Vis Sci 44: 217–225, for details on HSV-1 strain and virus stock preparation.
Ketamine hydrochloride Sigma K2753 Intraperitoneal injection of a solution containing Ketamine (100mg/kg) and Xylazine (10mg/kg)
Xylazine hydrochloride Sigma X1251
Paraformaldehyde (PFA) Sigma 158127 Suspend 4g of PFA in 90mL of water. Add 50µL of 1N NaOH, and heat at 60°C in a water bath with agitation. PFA dissolves in about 30min. Add 10mL of 10X PBS. This solution can be prepared in advance and stored at -20 °C in 5mL tubes. Caution. Manipulate under a fume hood.
Physiological Saline Sigma 07982-100TAB-F
1X PBS, pH 7.4 (sterile) Life Technologies 10010-015
Sucrose Sigma 84100 Prepare a 20% sucrose solution in 1X PBS.
Cryosectionning embedding medium – Tissue-Tek OCT Compound – SAKURA 4583
Large vector DNA purification kit Qiagen 12462 To purify Cosmid or BAC vector containing HSV-1 genome and store at -20°C
Nick translation kit Roche Applied Sciences 10 976 776 001
Cy3-dCTP GE Healthcare PA53021 Protect from light
0.5M EDTA Sigma E6758
G50 Mini spin column GE Healthcare 27-5330-01
Salmon sperm DNA 10mg/mL Invitrogen / Life Technologies 15632-011
Ethanol molecular biology grade Sigma 87047 Prepare a 70% solution
Salmon sperm DNA Invitrogen / Life Technologies 15632-011
Formamid Molecular biology grade Sigma F9037 Caution. Manipulate under fume hood.
HSV-1 biotinylated commercial probe Enzo Life Sciences ENZ-40838
ImmEdge hydrophobic pen Vector Laboratories H-4000
20X Saline Sodium Citrate (SSC) Sigma S6639 Prepare a 2X SSC solution in ddH20.
Triton X-100 Sigma T8787 Prepare a 10% stock solution in water and store at +4°C. Prepare the 0.5% solution in 1X PBS right before use.
10mM sodium citrate pH 6.0 Sigma S1804 Prepare a 100mM stock solution (10X). Weigh 10,5g of citric acid (MW 210.14. Caution, irritant and toxic, wear appropriate mask and gloves), and dissolve in 400mL water. Adjust pH at 6.0 with 1N NaOH (caution, irritant, wear gloves). Adjust to 500mL with distilled water. Dilute 10 times in distilled water before use.
Acetic Acid Sigma 320099
Methanol, molecular biology grade Sigma 322415
Dextran sulfate – MW 500 000 Euromedex EU0606-A
Denhardt's solution (100X) Euromedex 1020-A
Rubber Cement "FixoGum" Marabut 290110000
DNA purification kit – Qiaquick PCR purification kit – Qiagen 28104
T7 in vitro transcription kit Ambion / Life Technologies AM1314
Biotin-16-UTP Roche Applied Sciences 11388908910
RNA purification mini-column Qiagen 73404
Ribonucleoside Vanadyl Complex New England Biolabs S1402S
H2O2 Sigma H3410 Prepare a 3% solution in distilled water. Store at +4 °C and protect from light.
Yeast tRNA Invitrogen 15401011 prepare a 10mg/mL solution in RNAse free water
Normal Goat Serum Invitrogen PCN5000
Primary antibodies Any supplier The following primary antibodies were used in the result section: anti-mouse CENP-A (rabbit mAb C51A7, Cell Signaling Technologies), and anti-ATRX H-300 (Santa Cruz Biotechnology)
Secondary fluorescent antibodies Invitrogen / Life Technologies The fluorescent secondary antibodies routinely used in our protocol are AlexaFluor labeled goat antibodies (IgG H+L). The antibody used in the result section is an anti-rabbit goat antibody labaled with AlexaFluor 488 (reference A11001)
Tyramide Signal Amplification (TSA) kit – Streptavidin + AlexaFluor 350 (blue fluorescence) Invitrogen / Life Technologies #T20937 TSA kits are also available from Perkin Elmer
Tyramide Signal Amplification (TSA) kit – Streptavidin + AlexaFluor 488 (green fluorescence) Invitrogen / Life Technologies #T20932
Hoechst 33342 Invitrogen / Life Technologies H3570 Prepare a 0.5µg/mL solution in 1X PBS immediatly before use. Discard the remaining solution.
22x50mm coverslip. n°1.5 glass. Electron Microscopy Sciences 72204-04
Mounting medium with anti-fading agent – Vectashield – Vector Laboratories H-1000 Another conventional product is Fluoromount G from electron microscopy Science
Superfrost glass slides FisherScientific 12-550-15
EQUIPMENT
Equipment / material Company Reference Note
Needle for infection Glass micropipette hot drawn. Home made.
Dissection equipement Moria, France Microsurgical scissors and forceps
Peristaltic pump Cole Palmer Instruments Easyload Masterflex
Micro-syringe pump device (Nano Pump) kdScientific KDS310
Cryostat Leica France CM 1510-1
-80 °C freezer Sanyo Ultra Low -80°C
Domestic microwave oven
Dry block heater Eppendorf 022670204
Incubator Slide moat Boekel Scientific 240000
Coplin Jar Dominique Dutscher 68512
Staining glass container Dominique Dutscher 68506
Fluorescent microscope Zeiss The images presented in the result section were collected with a Zeiss AxioObserver with objective x40 LD NeoFluor N.A 0.6, and x100 PlanApochromat N.A 1.3. Filter set #38, #43 and #43. HXP 120 fluorescence light source. Photometrics CoolSNAP HQ2 CCD camera. Signal will be more easily observed on a recent high efficiency microscope such as Zeiss AxioImager/AxioObserver series, Nikon Ti-E/Ni-E series or Leica DM/DMI6000 series

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Catez, F., Rousseau, A., Labetoulle, M., Lomonte, P. Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining. J. Vis. Exp. (83), e51091, doi:10.3791/51091 (2014).

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