用于黑腿蜱、肩突硬蜱基因编辑的胚胎注射技术
Summary
本协议描述了一种注射蜱虫胚胎的方法。胚胎注射是遗传操作产生转基因系的首选技术。
Abstract
蜱虫可以传播各种病毒、细菌和原生动物病原体，因此被认为是具有医学和兽医重要性的载体。尽管蜱传疾病的负担越来越大，但由于将遗传转化工具应用于功能研究蜱的独特生物学的挑战，对蜱虫的研究仍然落后于昆虫病媒。遗传干预在减少蚊媒疾病方面越来越受到关注。然而，这种干预措施的发展需要通过注射胚胎来稳定地转化种系。螯合物（包括蜱虫）缺乏这种胚胎注射技术。几个因素，例如蜱胚胎上的外部厚蜡层、硬绒毛膜和高卵内压力，是以前阻碍蜱虫胚胎注射方案发展的一些障碍。目前的工作已经克服了这些障碍，这里描述了一种用于黑腿蜱（ 肩突硬 蜱）的胚胎注射技术。该技术可用于递送组分，如CRISPR/Cas9，以实现稳定的种系转化。
Introduction
蜱是具有医学和兽医重要性的载体，能够传播各种病毒、细菌、原生动物病原体和线虫1，2。在美国东部，黑腿蜱，肩突硬蜱，是莱姆病（LD）病原体伯氏螺旋体伯氏疏螺旋体的重要载体。美国每年报告的LD病例超过40万例，使其成为美国最大的媒介传播传染病1。除伯氏双歧杆菌外，肩突双歧杆菌还传播其他六种微生物，包括四种细菌（嗜吞噬细胞无形体、蛋黄孢杆菌、宫本双歧杆菌和穆氏埃立克体）、一种原生动物寄生虫（小巴贝虫）和一种病毒（波瓦桑病毒），使这种蜱虫物种成为主要的公共卫生问题3 .虽然近年来蜱传疾病变得越来越普遍，但由于蜱的独特生物学以及与应用遗传和功能基因组工具相关的挑战，对蜱虫的研究已经落后于其他节肢动物媒介，如蚊子4，5。
基因编辑技术，特别是CRISPR / Cas9，现在已经使功能基因组学研究在非模式生物中变得可行。为了在生物体中产生可遗传的突变，胚胎注射仍然是递送改变种系6，7，8，9的构建体的首选方法。然而，直到最近 4，蜱虫卵被认为太难甚至不可能在不杀死胚胎的情况下注射10，11。鸡蛋上厚厚的蜡层、坚硬的绒毛膜和高卵内压力是阻止蜱虫胚胎注射的一些主要障碍。成年，血饲肩胛骨I.在3-4周内沉积一窝多达2，000个卵12（约100个卵/天）。卵单独产下，每个卵都涂有蜡，蜡是由母亲的腺体器官13，14，15的突起或“角”分泌的。这种蜡可以保护鸡蛋免受干燥，并含有抗菌化合物15。为了成功注射蜱虫卵，重要的是要去除蜡层，软化绒毛膜，并使卵干燥以降低卵内压力，以使注射不会不可逆转地损害卵。了解胚胎注射对成功种系转化的至关重要性，开发了肩胛弧菌方案，可用于递送 CRISPR/Cas9 构建体并产生稳定的种系突变4.除了对肩胛蜱研究的贡献外，该协议还可以针对其他蜱物种进行优化。
Protocol
Representative Results
Discussion
这是第一个成功注射早期蜱虫胚胎的方案。已经实现了~4%-8%的存活率，这与其他成熟的昆虫模型中的胚胎注射相当5。
由于这是最初的协议，预计该协议将进一步完善并专门针对单个蜱物种。特别是，注射时间因物种而异，取决于胚胎发生，尤其是细胞化的时间。初步数据表明， 肩胛骨I. 卵在产卵后的前24小时内不会经历快速核分裂，几天后发生细胞?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢Channa Aluvihare和Yonus Gebermicale，ITF，UMD在协议开发的初始阶段提供的见解和支持。钨针是David O’Brochta，ITF，UMD的慷慨礼物。我们感谢Ladislav Simo博士在 I. ricinus 中测试了该协议，并进行了有见地的讨论。该项目由NIH-NIAID R21AI128393和纽约普利茅斯山基金会资助至MG-N，内华达大学向AN提供启动资金，国家科学基金会对MG-N和AN的资助编号2019609，以及IGTRCN向AS提供的点对点资助。
Materials
| Aluminum silicate capillaries, with filament | Sutter instruments | AF100-64-10 | Embryo injection |
| Benzalkonium chloride 50% in water, 25 g | TCI-America | B0414 | Embryo treatment, 25 g is approximately 25 mL |
| Filter paper | Whatman | 1001-090 | Post-injection care |
| Forceps | Thomas Scientific | 300-101 | Gene`s organ manipulation |
| Lab Wipes | Genesee Scientific | 88-115 | |
| Microloader tips | Eppendorf | 930001007 | Loading the pulled needles |
| Micromanipulator | Sutter instruments | ROE-200 | Embryo injection |
| Microscopic slides- plain, ground edges | Genesee Scientific | 29-100 | Embryo alignment, ground edges are preferred, beveled edges could obscure the eggs from view |
| NaCl | Research Products International | S23020-500.0 | Embryo treatment |
| Needle Puller | Sutter Instruments | P-1000 | |
| Permanent Double sided tape | Scotch | 34-8716-3417-5 | Embryo alignment |
| Petri plates | Genesee Scientific | 32-107G | Post-injection care |
| Tegaderm/ Transparent film dressing | 3M Healthcare | 1628 | Embryo alignment |
| Tungsten needles | Fine Science Tools | 10130-10 | Gene`s organ manipulation |
| Tungsten Wire | Amazon | B08DNT7ZK3 | Gene`s organ manipulation |
| XenoWorks Digital Microinjector | Sutter instruments | MPC-200 | Embryo injection |
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