冈 比亚按蚊 胚胎的显微注射方法
Summary
显微注射技术对于将外源基因引入蚊子的基因组至关重要。该协议解释了詹姆斯实验室使用的一种方法，该方法将DNA构建体微注射到 冈比亚按蚊 胚胎中以产生转化的蚊子。
Abstract
胚胎显微注射技术对于许多昆虫物种的分子和遗传学研究至关重要。它们提供了一种以稳定和可遗传的方式将编码感兴趣基因的外源DNA片段以及有利性状引入昆虫种系的方法。由此产生的转基因菌株可以研究由整合DNA的表达引起的表型变化，以回答基本问题或用于实际应用。虽然该技术很简单，但它需要调查人员的耐心和实践，以实现最大化效率的技能水平。这里展示的是一种对非洲疟疾蚊子冈 比亚按蚊胚胎进行显微注射的方法。目标是通过显微注射外源DNA递送到胚胎中，以便它可以在发育中的种系（极点）细胞中被吸收。转座酶、整合酶、重组酶或其他核酸酶（例如CRISPR相关蛋白，Cas）的注射DNA的表达可以触发导致其共价插入染色体的事件。由这些技术产生的转基因 冈比亚锥虫 已被用于免疫系统成分，参与血液喂养的基因和嗅觉系统元素的基础研究。此外，这些技术已被用于生产 冈比亚锥虫 菌株，其性状可能有助于控制疟疾寄生虫的传播。
Introduction
自20世纪初以来，显微注射技术已被用于实验性地操纵生物体1。显微注射已用于研究基本生物学功能和/或在所需生物体的生物学中引入重要变化。显微注射技术一直受到载体生物学家的特别兴趣，并已被广泛用于操纵载体基因组2-11。节肢动物载体中的转基因实验通常旨在通过实施降低载体适应性或增加对它们传播的病原体的难治性的改变来降低载体传播病原体的效率。蚊子传播多种人类病原体，对全世界的发病率和死亡率产生重大影响。蚊子的按蚊属传播人类疟疾寄生虫病原体疟原虫属。按蚊的基因工程实验旨在更好地了解这些蚊子的生物学并降低其媒介能力，以制定新的疟疾消除策略。
全世界疟疾感染最多的蚊媒位于 冈比亚按蚊 物种群中。然而，大多数成功的转基因实验都是在印度次大陆的疟疾病媒 按蚊斯蒂芬西上进行的。虽然存在大量实验室适应的 冈比亚按蚊 菌株，但文献中报道的转基因 冈比亚按蚊属 系的数量与 按蚊斯蒂芬西的菌株无法相比。据认为， 冈比亚按蚊 胚胎比 按蚊更难注射并实现成功的转基因，尽管这些差异的原因尚不清楚。该协议描述了一种已被证明通过显微注射实现 冈比亚按蚊 胚胎转基因遗传的方法。该协议基于Hervé Bossin和Mark Benedict12 先前开发的方法，并添加了一些额外的细节和更改，这些细节和更改已被发现可以提高转基因的效率。
Protocol
Representative Results
Discussion
随着CRISPR/Cas9等精确和灵活的基因工程技术的可用性增加，转基因生物体可以比以前更直接、更稳定地开发。这些工具使研究人员能够创造出蚊子媒介的转基因菌株，这些菌株非常接近于实现对病原体的难耐性（种群改变）或可遗传不育（种群抑制）的理想特性。然而，为了开发最安全、最稳定的转基因蚊子，需要创建和评估更多的转基因系，以便为实地研究选择最佳品系。所描述的方案可以提?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Drusilla Stillinger，Kiona Parker，Parrish Powell和Madeline Nottoli的蚊子饲养。资金由加州大学欧文分校疟疾倡议提供。AAJ是加州大学欧文分校的Donald Bren教授。
Materials
| 10x Microinjection Buffer | – | – | 1 mM NaHPO4 buffer, pH 6.8, 50 mM KCl |
| Blotting membrane (Zeta-Probe GT Genomic Tested Blotting Membrane) | Bio-Rad | Neatly and straightly cut into 2×1 cm piece | |
| Conical tubes 50 ml (disposable centrifuge tube, polypropylene) | Fisher Brand | Ends cut | |
| De-ionized or double-distilled water (ddH20) | Mili-Q | In a wash bottle | |
| Dissecting microscope | Leica | Leica MZ12 | For embryo alignment |
| Forceps | No. 5 size | ||
| Glass container | Pyrex | No. 3140 | 125 x 65 |
| Glass slide | Fisher Brand | No. 12-549-3 | 75×26 mm |
| Incubator | Barnsted Lab-line | Model No. 150 | 28 °C |
| KCl | 50 mM | ||
| Latex dental film | Crosstex International | No. 19302 | |
| Microinjector | Sutter Instrument | XenoWorks Digital Microinjector | |
| Microloader Pipette tips | Eppendorf | 20 µL microloader epT.I.P.S. | |
| Micromanipulator | Sutter Instrument | XenoWorks Micromanipulator | |
| Micropipette | Rainin | 20 µL | |
| Micropipette puller | Sutter Instrument | Sutter P-2000 micropipette puller | |
| Microscope | Leica | DM 1000 LED or M165 FC | For microinjection |
| Minimum fiber filter paper | Fisher Brand | No. 05-714-4 | Chromatography Paper, Thick |
| Mosquitoes | MR4, BEI Resources | Anopheles gambiae, mated adult females, blood-fed 4-5 days post-eclosion | |
| NaHPO4 buffer | 1 mM, ph 6.8 | ||
| Nylon mesh | |||
| Paint brush | Blick | No. 05831-7040 | Fine, size 4/0 |
| Petri dish | Plastic, (60×15 mm, 90×15 mm) | ||
| Sodium acetate | 3M | ||
| Quartz glass capillaries | Sutter Instrument | No. QF100-70-10 | With filament, 1 mm OD, ID 0.7 10 cm length |
| Water PCR grade | Roche | No. 03315843001 |
References
- Feramisco, J., Perona, R., Lacal, J. C., Lacal, J. C., Feramisco, J., Perona, R. Needle Microinjection: A Brief History. Microinjection. Methods and Tools in Biosciences and Medicine. , (1999).
- Windbichler, N., et al. A synthetic homing endonuclease-based gene drive system in the human malaria mosquito. Nature. 473 (7346), 212-215 (2011).
- Meredith, J. M., et al. Site-specific integration and expression of an anti-malarial gene in transgenic Anopheles gambiae significantly reduces Plasmodium infections. PLoS One. 6 (1), 14587 (2011).
- Hammond, A., et al. CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nature Biotechnology. 34 (1), 78-83 (2016).
- Carballar-Lejarazu, R., et al. Next-generation gene drive for population modification of the malaria vector mosquito, Anopheles gambiae. Proceedings of the National Academy of Sciences of the United States of America. 117 (37), 22805-22814 (2020).
- Simões, M. L., et al. The Anopheles FBN9 immune factor mediates Plasmodium species-specific defense through transgenic fat body expression. Develomental & Comparative Immunology. 67, 257-265 (2017).
- Arik, A. J., et al. Increased Akt signaling in the mosquito fat body increases adult survivorship. FASEB Journal. 4, 1404-1413 (2015).
- Riabinina, O., et al. Organization of olfactory centres in the malaria mosquito Anopheles gambiae. Nature Communications. 7, 13010 (2016).
- Kyrou, K., et al. A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes. Nature Biotechnology. 36 (11), 1062-1066 (2018).
- Dong, Y., Simões, M. L., Dimopoulos, G. Versatile transgenic multistage effector-gene combinations for Plasmodium falciparum suppression in Anopheles. Science Advances. 6 (20), (2020).
- Grossman, G. L., et al. Germline transformation of the malaria vector, Anopheles gambiae, with the piggyBac transposable element. Insect Molecular Biology. 6, 597-604 (2001).
- Benedict, M. Q. Methods in Anopheles research. Chapter 3: Specific Anopheles techniques. 3.1 Embryonic Techniques. 3.1.1 Microinjection methods for Anopheles Embryos. BEI resources. , (2015).
- Pham, T. B., et al. Experimental population modification of the malaria vector mosquito, Anopheles stephensi. PLoS Genetics. 15 (12), 1008440 (2019).
- Benedict, M. Q., et al. Pragmatic selection of larval mosquito diets for insectary rearing of Anopheles gambiae and Aedes aegypti. PLoS One. 15 (3), 0221838 (2020).
- Carballar-Lejarazú, R., James, A. A. Population modification of Anopheline species to control malaria transmission. Pathogens and Global Health. 111 (8), 424-435 (2017).
