壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫
Summary
这里，我们描述了通过使用脱乙酰壳多糖的抑制病媒蚊基因功能/干扰是由幼虫摄取的RNA纳米颗粒的过程。
Abstract
蚊子造成更多人的痛苦比任何其他生物–和杀死每年有超过百万人。蚊子基因组计划推动研究蚊子生物学的新方面，包括在初级非洲疟蚊冈比亚按蚊和登革热和黄热病载体埃及伊蚊功能基因研究。核糖核酸干扰 – （RNAi-）介导的基因沉默已用于靶向目的基因在这两种疾病的载体蚊子物种。在这里，我们描述了一个程序，制备壳聚糖/干扰RNA纳米颗粒相结合，与食物和幼虫摄取。这种技术上简单，高通量，并且相对便宜的方法，这是与长双链RNA（dsRNA）的或小的干扰RNA（siRNA）分子相容，已经使用了若干在A中不同基因的成功击倒冈比亚和A.伊蚊幼虫。以下幼虫喂食，击倒，这是通过定量RT-PCR或原位杂交证实，可以持续至少通过后期蛹期。这种方法可以适用于各种各样的蚊子和其它昆虫物种，包括农业害虫，以及其它非模式生物。除了其在研究实验室工具，在未来，脱乙酰壳多糖，一种廉价，无毒和可生物降解的聚合物，有可能被利用在该领域。
Introduction
在按蚊和Aedine属血液喂养蚊子传播负责几个人类的祸害最严重的致病因子。据估计，3.4十亿人的风险感染疟疾，负责每年全球有超过一半万人死亡。从感染疟疾结果由疟原虫属寄生虫，这是通过按蚊属感染的蚊子，包括主要的非洲病媒冈比亚按蚊 （叮咬传播给人类http://www.who.int/topics/malaria/en/ ，2014）1。 埃及伊蚊是登革热病毒，导致登革热，一种非特异性发热性疾病是世界上最普遍和显著虫媒病毒病的主要蚊子。登革热病毒是目前为> 2.5十亿人在热带地区的威胁，与每年inciden每年24000人死亡导致大约〜50000000案件CE（ http://www.cdc.gov/dengue/ ，2014）2。尽管蚊子传播的疾病，对人体健康的毁灭性的全球影响，预防和治疗这些疾病的有效手段缺乏。灭蚊是目前预防疾病的最佳方法。
用于控制通过媒介昆虫的遗传操作节肢动物传播的疾病的可能性已经认识超过四十年3。 A.转基因品系伊蚊设计有一个阻遏女性特有的飞型近期取得的潜力，利用转基因矢量控制策略成为现实4-6。这些进步都质疑研究人员确定了矢量控制和蚊子操纵基因功能的补充手段新的基因目标。基因表达ð维修uring发展，是女性飞蚊4的情况下，可能会促进新的矢量控制策略的阐释。但是，这主要是由于技术上的挑战，很少基因的功能已被鉴定A.发展过程中冈比亚，埃及伊蚊，或其他蚊种。
自从发现C.线虫 7，RNA干扰（RNAi），这是保守的动物，植物和微生物，已被广泛用于功能基因研究中的各种生物，包括昆虫8,9。 RNAi途径由Dicer酶，它劈开长双链RNA进入开始充当序列特异性RNA干扰短20-25个核苷酸长的siRNA。的siRNA沉默基因是在互补序列通过促 进转录周转，裂解和翻译9的中断。长的dsRNA分子（通常为300-600碱基对）或定制的siRNA靶向particulař序列可用于在研究实验室用于沉默感兴趣的任何基因。当通过RNA干扰交付管理，研究人员可以控制时间，其中的基因沉默发起。因为它可以用来克服的挑战，如发育杀伤力或无菌，可阻碍生产和维护菌株轴承可遗传突变，一个昂贵的和劳动密集的过程，目前尚未在所有昆虫种类可用的这个优点是非常有用的。虽然基因沉默的RNA干扰的程度可以从基因而异基因，组织对组织和动物动物，RNAi技术被广泛地用于基因蚊子和其它昆虫8,9的功能分析。
三个干扰RNA递送策略已用于蚊子：显微注射，浸泡/局部施用，并摄取。进行了详细的历史和在昆虫中使用这三种技术中的比较，请参阅Yu 等人 8。 W¯¯E具有成功地使用显微注射10作为递送的siRNA的一种手段，以靶向A.发育基因伊蚊胚胎，幼虫，蛹和11-14。然而，这种劳动密集型的投放策略同时需要显微注射的设置和熟练的手。此外，显微注射是压力的生物体，一个混杂因素，特别是当行为表型进行评估。最后，显微注射递送不能扩展到田间进行矢量控制。作为替代，浸泡在干扰RNA溶液中的生物也成为一种流行的诱导基因沉默的手段，因为它是方便的，并且需要很少的设备或劳力。浸泡已经主要是在昆虫细胞系被应用研究8，但在最近的一项研究，在击倒A.达到伊蚊幼虫浸入dsRNA的15，其中动物似乎是摄取的溶液。然而，对于涉及多个experimenta的分析研究升组或表型，浸泡是相当昂贵的。洛佩兹-马丁内斯等人 16描述补液驱动的RNAi，一种新的方法来干扰RNA交付，涉及脱水生理盐水补液和用含有RNA干扰一滴水。这种方法并切断与整个动物浸没相关的成本，但是比注射更昂贵，并且可以将其应用到可容忍高渗透压物种的限制。此外，它是很难设想如何浸没或脱水/再水化浸渍方法可适于在磁场矢量控制。由于这些原因，用于后胚胎的研究，递送干扰RNA与摄入食物是一种可行的替代策略。
虽然摄入为基础的战略并不适用于所有的昆虫种类，也许最值得注意的是果蝇 ，口服给药干扰RNA与食物混合促进基因SIlencing在各种昆虫8,17，包括A.伊蚊成年人18。我们在A.描述壳聚糖纳米介导的RNA干扰冈比亚幼虫19，并已成功地应用这种方法为A.降低基因表达伊蚊幼虫20,21。这里，方法为这个RNAi的过程，这涉及截留通过聚合物壳聚糖干扰RNA的，是详尽。脱乙酰壳多糖/干扰RNA纳米颗粒通过聚阳离子的自组装通过氨基的脱乙酰壳多糖中的正电荷和由磷酸基团上的干扰RNA 19的骨干承载负电荷之间的静电引力干扰RNA形成。所述的程序是与两个长的dsRNA分子（以下简称为双链RNA）或双链的siRNA（以下简称为干扰）兼容。以下的合成，脱乙酰壳多糖/干扰RNA纳米颗粒混合，幼虫的食物，并传送到幼虫通过口服。这种方法相对便宜，只需要很少的设备和劳动力19，并有利于多种表型，包括行为20,21的分析的高通量分析。这种方法，可以适用于在其他昆虫，包括其它疾病的载体和昆虫农业害虫的基因沉默研究，有可能在各种其他动物物种的用于基因沉默。此外，脱乙酰壳多糖，一种廉价，无毒，可生物降解的聚合物22，有可能被利用在该领域为物种特异性蚊虫控制。
Protocol
Representative Results
Discussion
脱乙酰壳多糖/干扰RNA纳米颗粒本文所描述的方法已被用于在A中幼虫发育期间有效地针对基因冈比亚 （ 图2,3）和A.埃及伊蚊 （ 图4，5，6，表1，表2）。壳聚糖的纳米颗粒可以与任一长的dsRNA或siRNA，这两者已被成功地用于在蚊子就证明了这里所描述的代表性的结果来制备。 dsRNA的合成比购买的siRNA更便宜，但更耗费人力。如果脱乙酰壳多糖/ siRNA的被使?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Funding from the Kansas Agricultural Experiment Station and K-State Arthropod Genomics Center to KYZ supported the original development of chitosan/dsRNA-mediated gene silencing in A. gambiae19.The A. gambiae work described here was supported in part by R01-AI095842 from NIH/NIAID to KM. Development of chitosan/siRNA-mediated gene silencing in A. aegypti20,21 was supported by NIH/NIAID Award R01-AI081795 to MDS. The contents of this study are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
Materials
| Name of Reagent/Equipment | Company | Catalog Number | Comments/Descriptions |
| 0.02 ml pipetteman | Rainin | PR20 | for resuspension of interfering RNA |
| 0.2 ml pipetteman | Rainin | PR200 | for resuspension of interfering RNA and preparation of interfering RNA/nanoparticles |
| 0.5 ml graduated tube | Fischer Scientific | 05-408-120 | for aliquoting resuspended interfering RNA |
| 1 ml pipetteman | Rainin | PR1000 | for resuspension of interfering RNA and preparation of interfering RNA/nanoparticles |
| 1.5 ml graduated tube | Fischer Scientific | 05-408-046 | for preparation of interfering RNA/nanoparticles |
| 1M Sodium Acetate, pH 4.5 | TEKnova | S0299 | to prepare sodium acetate buffer |
| Acetic Acid, AIRSTAR. ACS, USP, FCC Grade | BDH | VWR BDH3092-500MLP | to prepare sodium acetate buffer |
| Agarose Genetic Technology Grade | MP Biomedicals LLC. | 800668 | to coat prepared interfering RNA/nanoparticles |
| Centrifuge | Eppendorf AG | 5415D | to pellet interfering RNA/nanoparticles |
| Chitosan, from shrimp shells | Sigma-Aldrich | C3646-25G | to combine with interfering RNA for prepararation of interfering RNA/nanoparticles |
| Dry Yeast | Universal Food Corp | NA | to prepare mosquito larval food with interfering RNA/nanoparticles |
| E-RNAi tool | German Cancer Research Center | NA | for design of dsRNA; http://www.dkfz.de/signaling/e-rnai3// |
| goldfish food | Wardley | Goldfish FLAKE FOOD | to prepare mosquito larval food with interfering RNA/nanoparticles |
| Heated water bath | Thermo Scientific | 51221048 | to heat the interfering RNA/nanoparticles at 55oC |
| Ice | not applicable | not applicable | for thawing interfering RNA and preparation of interfering RNA/nanoparticles |
| Ice bucket | Fisher Scientific | 02-591-44 | for storage of ice used during the procedure |
| liver powder | MP Biomedicals LLC. | 900396 | to feed mosquito larvae post interfering RNA/nanoparticle treatment |
| Microwave oven | A variety of vendors | not applicable | to prepare 2% agarose solution |
| petridish (100X15 mm) | Fischer Scientific | 875713 | interfering RNA/nanoparticle feeding chamber for larvae |
| pH meter | Mettler Toledo | S220 | for preparation of buffers |
| Razor blade | Fischer Scientific | 12-640 | to divide the interfering RNA/nanoparticle pellet for feedings |
| siRNA | Thermo Scientific/Dharmacon | custom | for preparation of siRNA/nanoparticles |
| Sodium Sulfate, Anhydrous (Na2SO4) | BDH/distributed by VWR | VWR BDH0302-500G | to prepare 50mM sodium sulfate solution in which the interfering RNA will be resuspended |
| Thermometer | VWR | 61066-046 | to measure the water bath temperature |
| Tooth picks | VWR | 470146-908 | for stirring during interfering RNA/nanoparticle food preparation and cutting gel pellets |
| Ultralow freezer | A variety of vendors | not applicable | for storage of interfering RNA aliquots and interfering RNA/nanoparticles at -80oC |
| Vortex mixer | Fischer Scientific | 02-216-108 | for preparation of chitosan/interfering RNA |
| Weight paper | Fischer Scientific | NC9798735 | to divide the interfering RNA/nanoparticle pellet for feedings |
References
- Knipling, E. F., et al. Genetic control of insects of public health importance. Bulletin of the World Health Organization. 38 (3), 421-438 (1968).
- Fu, G., et al. Female-specific flightless phenotype for mosquito control. Proceedings of the National Academy of Sciences of the United States of America. 107 (10), 4550-4554 (2010).
- Wise de Valdez, M. R., et al. Genetic elimination of dengue vector mosquitoes. Proceedings of the National Academy of Sciences of the United States of America. 108 (12), 4772-4775 (2011).
- Harris, A. F., et al. Successful suppression of a field mosquito population by sustained release of engineered male mosquitoes. Nature Biotechnology. 30 (9), 828-830 (2012).
- Fire, A., et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 391 (6669), 806-811 (1998).
- Yu, N., et al. Delivery of dsRNA for RNAi in insects: an overview and future directions. Insect science. 20 (1), 4-14 (2013).
- Zhang, H., Li, H. C., Feasibility Miao, X. X. limitation and possible solutions of RNAi-based technology for insect pest control. Insect science. 20 (1), 15-30 (2013).
- Clemons, A., Haugen, M., Severson, D. W., Duman-Scheel, M. Functional analysis of genes in Aedes aegypti embryos. Cold Spring Harb Protoc. 2010 (10), (2010).
- Clemons, A., et al. siRNA-mediated gene targeting in Aedes aegypti embryos reveals that frazzled regulates vector mosquito CNS development. PLoS One. 6 (1), e16730 (2011).
- Haugen, M., et al. Semaphorin-1a is required for Aedes aegypti embryonic nerve cord development. PLoS One. 6 (6), e21694 (2011).
- Nguyen, C., et al. Functional genetic characterization of salivary gland development in Aedes aegypti. EvoDevo. 4 (1), 9 (2013).
- Sarro, J., et al. Requirement for commissureless2 function during dipteran insect nerve cord development. Developmental dynamics: an official publication of the American Association of Anatomists. 242 (12), 1466-1477 (2013).
- Singh, A. D., Wong, S., Ryan, C. P., Whyard, S. Oral delivery of double-stranded RNA in larvae of the yellow fever mosquito, Aedes aegypti: implications for pest mosquito control. J Insect Sci. 13, 69 (2013).
- Lopez-Martinez, G., Meuti, M., Denlinger, D. L. Rehydration driven RNAi: a novel approach for effectively delivering dsRNA to mosquito larvae. Journal of medical entomology. 49 (1), 215-218 (2012).
- Scott, J. G., et al. Towards the elements of successful insect RNAi. Journal of insect physiology. 59 (12), 1212-1221 (2013).
- Coy, M. R., et al. Gene silencing in adult Aedes aegypti mosquitoes through oral delivery of double-stranded RNA. J Appl Entomol. 136 (10), 741-748 (2012).
- Zhang, X., Zhang, J., Zhu, K. Y. Chitosan/double-stranded RNA nanoparticle-mediated RNA interference to silence chitin synthase genes through larval feeding in the African malaria mosquito (Anopheles gambiae). Insect molecular biology. 19 (5), 683-693 (2010).
- Mysore, K., Flannery, E. M., Tomchaney, M., Severson, D. W., Duman-Scheel, M. Disruption of Aedes aegypti olfactory system development through chitosan/siRNA nanoparticle targeting of semaphorin-1a. PLoS neglected tropical diseases. 7 (5), e2215 (2013).
- Mysore, K., Andrews, E., Li, P., Duman-Scheel, M. Chitosan/siRNA nanoparticle targeting demonstrates a requirement for single-minded during larval and pupal olfactory system development of the vector mosquito Aedes aegypti. BMC developmental biology. 14 (1), 9 (2014).
- Dass, C. R., Choong, P. F. Chitosan-mediated orally delivered nucleic acids: a gutful of gene therapy. Journal of drug targeting. 16 (4), 257-261 (2008).
- An, C., Budd, A., Kanost, M. R., Michel, K. Characterization of a regulatory unit that controls melanization and affects longevity of mosquitoes. Cellular and molecular life sciences : CMLS. 68 (11), 1929-1939 (2011).
- Clemons, A., Mori, A., Haugen, M., Severson, D. W., Duman-Scheel, M. Culturing and egg collection of Aedes aegypti. Cold Spring Harb Protoc. 2010 (10), (2010).
- Horn, T., Boutros, M. E-RNAi: a web application for the multi-species design of RNAi reagents–2010 update. Nucleic acids research. 38, W332-W339 (2010).
- Patel, N. H., PA, K. r. i. e. g. Ch. 19. In situ hybridization to whole mount Drosophila embryos. A laboratory guide to RNA: Isolation, Analysis, and Synthesis. , (1996).
- Haugen, M., et al. . Whole-mount in situ hybridization for analysis of gene expression during Aedes aegypti development. 2010 (10), (2010).
- Clemons, A., Flannery, E., Kast, K., Severson, D. W., Duman-Scheel, M. Immunohistochemical analysis of protein expression during Aedes aegypti development. Cold Spring Harb Protoc. 2010 (10), (1101).
- Rund, S. S., Hou, T. Y., Ward, S. M., Collins, F. H., Duffield, G. E. Genome-wide profiling of diel and circadian gene expression in the malaria vector Anopheles gambiae. Proceedings of the National Academy of Sciences of the United States of America. 108 (32), E421-E430 (2011).
- Gaj, T., Gersbach, C. A., Barbas, C. F. ZFN, TALEN, CRISPR/Cas-based methods for genome engineering. Trends in biotechnology. 31 (7), 397-405 (2013).
- Aryan, A., Myles, K. M., Adelman, Z. N. Targeted genome editing in Aedes aegypti using TALENs. Methods. 69 (1), 38-45 (2014).
- Sarathi, M., Simon, M. C., Venkatesan, C., Hameed, A. S. Oral administration of bacterially expressed VP28dsRNA to protect Penaeus monodon from white spot syndrome virus. Mar Biotechnol (NY). 10 (3), 242-249 (2008).
- He, B., et al. Fluorescent nanoparticle delivered dsRNA toward genetic control of insect pests). Adv Mater. 25 (33), 4580-4584 (2013).
- Giljohann, D. A., Seferos, D. S., Prigodich, A. E., Patel, P. C., Mirkin, C. A. Gene regulation with polyvalent siRNA-nanoparticle conjugates. Journal of the American Chemical Society. 131 (6), 2072-2073 (2009).
- Davis, M. E., et al. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature. 464 (7921), 1067-1070 (2010).
- Molinaro, R., et al. Polyethylenimine and chitosan carriers for the delivery of RNA interference effectors. Expert opinion on drug delivery. 10 (12), 1653-1668 (2013).
- Singha, K., et al. Polymers in small-interfering RNA delivery. Nucleic acid therapeutics. 21 (3), 133-148 (2011).
