粒状棘球绦虫的短暂转导
1Student Research Committee, School of Medicine,Kerman University of Medical Sciences, 2Research Center for Hydatid Disease in Iran,Kerman University of Medical Sciences, 3Department of Parasitology and Mycology, Faculty of Medicine,Kurdistan University of Medical Sciences, 4State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine,Jilin University
Summary
我们使用第三代慢病毒载体描述了粒状 棘球绦虫 不同发育阶段的快速瞬时转导技术。
Abstract
囊性棘球蚴病或包虫病是由 粒状棘球绦虫 引起的最重要的人畜共患寄生虫病之一，棘球绦虫是一种藏耙在犬科动物肠道中的小绦虫。迫切需要应用遗传学研究来了解发病机制和疾病控制和预防。然而，缺乏有效的基因评估系统阻碍了对囊寄生虫（包括 棘球绦虫 属）的功能遗传学的直接解释。本研究证明了慢病毒基因瞬时转导在 颗粒埃马氏菌的元切除和杂化形式中的潜力。从包虫囊肿中分离出原结肠（PSC）并转移到特定的双相培养基中以发育成蠕动的蠕虫。用收获的第三代慢病毒以及HEK293T细胞转染蠕虫作为转导过程对照。在24 h和48 h的蠕动蠕虫中检测到明显的荧光，表明 颗粒埃布氏菌的瞬时慢病毒转导。这项工作首次尝试了绦虫中基于慢病毒的瞬时转导，并展示了对扁虫生物学实验研究具有潜在意义的有希望的结果。
Introduction
囊性棘球蚴病（CE）是由 粒状棘球绦 虫引起的最重要的蠕虫疾病之一，颗粒棘球绦虫是绦虫科1，2中的一种小型绦虫。已经对 颗粒埃布氏菌 的免疫诊断和疫苗开发进行了广泛的研究。然而，对寄生虫生物学分子基础的了解不足对包虫病的诊断、管理和预防造成了重大限制3，4，5，6。
近年来，由于基因组测序和转录组学方法的发展，几个研究小组7，8，9对扁虫进行了广泛的分子研究。然而，在寄生虫世界中，与为某些原生动物10，11，12开发的高度可重复的瞬时转导方法相比，寄生扁虫的基因转移技术进展仍然有限。
在过去二十年中，病毒递送系统的使用已成为转基因递送和基因/蛋白质研究的重要工具13.慢病毒感染分裂和非分裂细胞，从而有可能感染有丝分裂后细胞14，15，16。最近的证据表明，在哺乳动物细胞中使用基于慢病毒的转导系统有可能克服以前敲入/敲低技术的大部分局限性。具有适当分子标记（例如GFP表达）的表达慢病毒载体的设计和构建在前面已经描述过16。因此，我们评估 了颗粒埃马氏菌原结肠和蠕动蠕虫中GFP报告基因的慢病毒瞬时转导。
Protocol
本研究由国家医学研究发展研究所和研究伦理审查委员会批准，编号：958680。慢病毒被归类为BSL-2生物;因此，该协议中的所有实验室培养程序均使用无菌实验室实践进行，并根据NIH指南在层流罩下进行。 图1 显示了不同 颗粒芽孢杆菌 阶段的研究方案的示意图。 1. 收集包虫囊肿 从屠宰场经常屠宰的自然感染的绵羊中收集肝脏?…
Representative Results
在这里，我们通过使用第三代慢病毒载体描述了颗粒桔梗的快速有效的瞬时转导技术。我们在双相培养基中培养PSCs以获得蠕动的蠕虫，如前所述25，26。原结肠在体外6周后发育成蠕动的蠕虫。在双相培养基中观察到颗粒杆菌的不同阶段，包括内陷的PSCs（图2A），蒸发的PSCs（图2B）和具有?…
Discussion
了解线虫和梧桐生物学的分子基础对于理解人畜共患寄生虫的致病性至关重要27.缺乏有效的基因评估系统是直接解释茴香寄生虫（包括 棘球绦虫 属12，27）功能性遗传学的主要障碍。本研究证明了慢病毒在 细粒埃布氏菌 瞬时转导中的优异潜力。
慢病毒转导将瞬时转导的简单使用和速度与稳定细胞系?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本出版物中报道的研究得到了精英研究人员资助委员会的支持，奖励编号为958680，来自伊朗德黑兰国家医学研究发展研究所（NIMAD）。
Materials
| 12-well culture plates | SPL Life Sciences | 30012 | |
| 25 cm2 culture flask | SPL Life Sciences | 70325 | |
| 6-well culture plates | SPL Life Sciences | 30006 | |
| Calcium chloride | Sigma-Aldrich | C4901-500G | Working concentration: 2.5 mM |
| CMRL 1066 medium | Thermo Fisher Scientific | 11530037 | |
| CO2 incubator | memmert | ICO150 | |
| D-(+)-Glucose | Sigma-Aldrich | G8270-1KG | |
| DMEM | Life Technology | 12100046 | |
| Dog bile | Isolated from a euthanized dog and sterilized by 0.2 μm syringe filter | ||
| Eosin Y | Sigma-Aldrich | E4009-5G | prepare 0.1% of Eosin for working exclusion test |
| Fetal Bovine Serum (FBS) | DNAbiotech | DB9723-100ml | Heat inactivation of FBS (30 min in 40 °C) |
| Fetal Bovine Serum (FCS) | DNAbiotech | DB9724-100ml | Heat inactivation of FCS (30 min in 40 °C) |
| HEK293T cells | BONbiotech | BN_0012.1.14 | Human embryonic kidney 293T |
| HEPES buffered saline (HBS) | Sigma-Aldrich | 51558-50ML | 2x concentrate |
| Inverted fluorescence microscope | OLYMPUS | IX51 | |
| Penicillin | Sigma-Aldrich | P3032-10MU | Working concentration: 100 IU/mL |
| Pepsin | Roche | 10108057001 | Working concentration: 2 mg/mL, pH 2 |
| Phosphate-buffered saline (PBS) | DNAbiotech | DB0011 | This reagent solve in less than 1 min in D.W |
| Polybrene (Transfection reagent) | Sigma-Aldrich | TR-1003-G | |
| RPMI medium | BioIdea | BI-1006-05 | |
| Sodium bicarbonate (NaHCO3) | Sigma-Aldrich | S5761-1KG | |
| Streptomycin | Sigma-Aldrich | S9137-25G | Working concentration: 100 μg/mL |
| Third-generation lentiviral plasmid (pCDH513b) | SBI System Biosciences (BioCat GmbH) | CD513B-1-SBI | Transfer vector (obtained commercially from Molecular Medicine Research Department of Iranian Academic Center for Education, Culture and Research (ACECR), Mashhad, Iran) |
| Third-generation lentiviral plasmid (pLPI and pLPII) | Invitrogen (Life Technologies) | K4975-00 | Helper vector (obtained commercially from Molecular Medicine Research Department of Iranian Academic Center for Education, Culture and Research (ACECR), Mashhad, Iran) |
| Third-generation lentiviral plasmid (pMD2G) | Addgene | Plasmid 12259 | Helper vector (obtained commercially from Molecular Medicine Research Department of Iranian Academic Center for Education, Culture and Research (ACECR), Mashhad, Iran) |
| Tris/EDTA Buffer (TE) | DNAbiotech | DB9713-100ml | |
| Trypsin | Sigma-Aldrich | T9935-50MG | 1x working solutions (pH 7.4–7.6) |
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Cite This Article
Mohammadi, M. A., Afgar, A., Faridi, A., Mousavi, S. M., Derakhshani, A., Borhani, M., Fasihi Harandi, M. Transient Transduction of the Strobilated Forms of Echinococcus granulosus. J. Vis. Exp. (187), e62783, doi:10.3791/62783 (2022).