纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达
Summary
在成年斑马鱼器官中发展有条件基因剔除或有效基因击倒仍是一项重大挑战。在这里, 我们报告了在成年斑马鱼心脏执行纳米粒子介导的 siRNA 基因沉默的协议, 从而为研究斑马鱼和其他模型生物体中的成年器官提供了一种新的功能损失方法。
Abstract
哺乳动物在心肌梗死后再生心脏的能力非常有限。另一方面, 成年斑马鱼在先端切除或 cryoinjury 后再生心脏, 使其成为心脏再生研究的重要模型机体。然而, 缺乏功能丧失的成人器官的方法, 限制了对心脏再生机制的洞察力。RNA 干扰通过不同的运载系统是一个强有力的工具为沉默基因在哺乳动物细胞和模型有机体。我们以前曾报道, siRNA 纳米粒子成功地进入细胞, 并导致在再生的成年斑马鱼心脏显着基因特定的击倒。在这里, 我们提出了一个简单, 快速, 有效的协议大分子介导的 siRNA 交付和基因沉默的再生成年斑马鱼心脏。该方法为确定斑马鱼成年器官中的基因功能提供了一种替代方法, 可推广到其他模型生物中。
Introduction
心肌梗死已成为严重的健康威胁, 导致1世界各地的巨大经济负担。成年哺乳动物心脏在损伤后无法再生和补充失去的心肌细胞, 导致瘢痕组织的形成和随后的心力衰竭。与哺乳动物不同的是, 斑马鱼能够心脏再生, 主要是通过各种类型的心脏损伤后的强健心肌增生, 使其成为研究心脏再生分子机制的理想模型有机体。2,3,4,5,6,7,8。破译斑马鱼心脏再生的内源机制是一个令人兴奋的研究领域, 寻找新的治疗策略, 以改善人类心脏再生9。
在斑马鱼身上有遗传操作方法。这些包括 morpholinos (MO), 也广泛用于青蛙, 小鸡和哺乳动物除斑马鱼10,11,12,13。MO 在成年斑马鱼鳍, 脑, 视网膜14,15,16,17,18,19, 有效地击倒靶基因表达。锁定核酸 (低噪声放大器) 是另一种用于除斑马鱼胚胎中的内源基因表达的人工寡核苷酸, 在成年动物器官中也有20,21,22,23,24. 然而, 对于成年人心脏缺乏有效的功能丧失方法, 仍然是研究器官再生分子机制的一个障碍。目前, 小分子抑制剂或显性阴性突变体的转基因表达主要用于阻断某一基因或通路的功能, 以研究其在斑马鱼心脏再生25、26 中的作用. ,27。然而, 并非所有的基因或信号通路都适用于这些方法。
小干扰 rna (siRNAs) 广泛应用于哺乳动物细胞和模型生物体胚胎的功能损失分析, 以及动物模型28、29、30的临床前研究的成人器官。,31,32. siRNAs 已有效地用于沉默的肿瘤33,34,35和心肌细胞36,37,38,39 的基因 ,40 通过不同的交付系统。最近, 我们开发了有效的 siRNA 封装纳米颗粒基因沉默的再生成人心脏使用几种不同的纳米粒子41,42,43, 提供了一个新的工具成年斑马鱼器官基因的功能研究。根据我们以前的研究41,42,43, 这里我们提出了一个简单的, 实用的, 但强大的协议 siRNA 基因沉默在再生成年斑马鱼心脏使用 f-PAMAM-PEG-R9大分子.Aldh1a2(醛脱氢酶1家族, 成员 A2) 基因上调后, 斑马鱼的先端切除和消融Aldh1a2阻断心脏再生44。以aldh1a2基因为例, 对纳米粒子包覆 siRNA 注射液介导的基因击倒效率进行了测试。本协议包含了斑马鱼心脏切除、纳米微粒的化学合成和 siRNA 包裹纳米微粒向成年斑马鱼心脏的传递方法。
Protocol
所有动物程序都使用了由北京大学机构动物护理和使用委员会批准的斑马鱼议定书, 该协议由实验室动物护理评估和认可协会完全认可。 1. Tricaine 溶液的制备 要准备 tricaine 的溶液, 添加400毫克 3-氨基苯甲酸甲基磺酸粉到97.9 毫升蒸馏水, 然后加入2.1 毫升的1米 (ph 9.5) 调整 ph 值 ~ 7。将库存解决方案存储在摄氏4摄氏度。 为准备 tricaine 工作解决方案, 添加4.2 毫升 tr…
Representative Results
为了确定大分子介导的 siRNA 分娩的效率, 我们切除了斑马鱼心脏心室的顶端, 然后注射了约10µL 的大分子 (模拟组), Cy5-siRNA 只 (裸组), 或 f-PAMAM-PEG-R9 大分子封装Cy5-siRNA (Cy5-siRNA 组) intrapleurally 分别 (图 2A B)。荧光信号是可检测的心脏注射大分子封装 Cy5-siRNA 在 3, 24 和 48 hpi (小时后注射), 而它是很难检测到心脏从模拟和裸组在 48 hpi (<strong cla…
Discussion
斑马鱼完全有能力再生各种器官, 包括成人心脏5。尽管转基因和遗传方法在研究斑马鱼胚胎中的基因功能方面发展良好, 研究者仍面临着在斑马鱼45,46中产生条件突变基因的艰巨任务。因此, 转基因显性阴性突变体或小分子抑制剂经常被用来处理成年斑马鱼器官的基因功能25,27,<sup class…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢 IC 布鲁斯博士的批评性评论和阅读手稿。这项工作得到了中国国家自然科学基金 (31430059、31701272、31730061、81470399和 31521062)、亚洲经济和新兴市场创新医学和早期发展的资助。
Materials
| tricaine | Sigma | E10521 | Store at 4°C |
| stereomicroscope | Leica | S8AP0 | |
| sharp forcep | WPI | 14098 | |
| iridectomy scissors | WPI | 501778 | |
| elbow tweezers | Suzhou Liuliu | SE05Cr | |
| α,ω-dipyridyl disulfido polyethylene glycol(Py-PEG-Py) | Biomatrik (Jiaxing) Inc. | 5239 | |
| core of G4.0 polyamidoamine (PAMAM) | Andrews ChemServices | AuCS-297 | |
| vacuum drying equipment | Yiheng | DZF-6020 | |
| Dulbecco's phosphate-buffered saline (DPBS) | Gibco | 14190144 | |
| tris(2-carboxyethyl)phosphine(TCEP) | Alfar Aesar | 51805-45-9 | Causes severe skin burns and eye damage. Causes serious eye damage. |
| ultrafiltration tube | Millipore | UFC900308 | |
| freeze dryer | Martin Christ | Alpha 2-4 Ldplus | |
| NMR spectrometer | Bruker | AV400 | |
| Deuterium oxide(D2O) | J&K | 174611 | |
| NMR sample tube | J&K | WG-1000-7-50 | |
| 3 kDa MWCO ultrafiltration tube | Merck | UFC900308 | |
| sea salts | Instant Ocean® | SS15-10 |
References
- Writing Group Members. Executive Summary: Heart Disease and Stroke Statistics–2016 Update: A Report From the American Heart Association. Circulation. 133 (4), 447-454 (2016).
- Chablais, F., Veit, J., Rainer, G., Jazwinska, A. The zebrafish heart regenerates after cryoinjury-induced myocardial infarction. BMC Dev Biol. 11, 21 (2011).
- Gonzalez-Rosa, J. M., Martin, V., Peralta, M., Torres, M., Mercader, N. Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish. Development. 138 (9), 1663-1674 (2011).
- Parente, V., et al. Hypoxia/reoxygenation cardiac injury and regeneration in zebrafish adult heart. PLoS One. 8 (1), 53748 (2013).
- Poss, K. D., Wilson, L. G., Keating, M. T. Heart regeneration in zebrafish. Science. 298 (5601), 2188-2190 (2002).
- Raya, A., et al. Activation of Notch signaling pathway precedes heart regeneration in zebrafish. Proc Natl Acad Sci U S A. 100, 11889-11895 (2003).
- Schnabel, K., Wu, C. C., Kurth, T., Weidinger, G. Regeneration of cryoinjury induced necrotic heart lesions in zebrafish is associated with epicardial activation and cardiomyocyte proliferation. PLoS One. 6 (4), 18503 (2011).
- Wang, J., et al. The regenerative capacity of zebrafish reverses cardiac failure caused by genetic cardiomyocyte depletion. Development. 138 (16), 3421-3430 (2011).
- Gonzalez-Rosa, J. M., Burns, C. E., Burns, C. G. Zebrafish heart regeneration: 15 years of discoveries. Regeneration (Oxf). 4 (3), 105-123 (2017).
- Heasman, J., Kofron, M., Wylie, C. Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. Dev Biol. 222 (1), 124-134 (2000).
- Nasevicius, A., Ekker, S. C. Effective targeted gene ‘knockdown’ in zebrafish. Nat Genet. 26 (2), 216-220 (2000).
- Coonrod, S. A., Bolling, L. C., Wright, P. W., Visconti, P. E., Herr, J. C. A morpholino phenocopy of the mouse mos mutation. Genesis. 30 (3), 198-200 (2001).
- London, C. A., et al. A novel antisense inhibitor of MMP-9 attenuates angiogenesis, human prostate cancer cell invasion and tumorigenicity. Cancer Gene Ther. 10 (11), 823-832 (2003).
- Kizil, C., Otto, G. W., Geisler, R., Nusslein-Volhard, C., Antos, C. L. Simplet controls cell proliferation and gene transcription during zebrafish caudal fin regeneration. Dev Biol. 325 (2), 329-340 (2009).
- Thummel, R., et al. Inhibition of zebrafish fin regeneration using in vivo. electroporation of morpholinos against fgfr1 and msxb. Dev Dyn. 235 (2), 336-346 (2006).
- Kizil, C., Brand, M. Cerebroventricular microinjection (CVMI) into adult zebrafish brain is an efficient misexpression method for forebrain ventricular cells. PLoS One. 6 (11), 27395 (2011).
- Kizil, C., Iltzsche, A., Kaslin, J., Brand, M. Micromanipulation of gene expression in the adult zebrafish brain using cerebroventricular microinjection of morpholino oligonucleotides. J Vis Exp. (75), e50415 (2013).
- Craig, S. E., et al. The zebrafish galectin Drgal1-l2 is expressed by proliferating Muller glia and photoreceptor progenitors and regulates the regeneration of rod photoreceptors. Invest Ophthalmol Vis Sci. 51 (6), 3244-3252 (2010).
- Thummel, R., Bailey, T. J., Hyde, D. R. In vivo electroporation of morpholinos into the adult zebrafish retina. J Vis Exp. (58), e3603 (2011).
- Rayburn, E. R., Zhang, R. Antisense, RNAi and gene silencing strategies for therapy: mission possible or impossible. Drug Discov Today. 13 (11-12), 513-521 (2008).
- Seth, P. P., et al. Short antisense oligonucleotides with novel 2′-4′ conformationaly restricted nucleoside analogues show improved potency without increased toxicity in animals. J Med Chem. 52 (1), 10-13 (2009).
- Prakash, T. P., et al. Antisense oligonucleotides containing conformationally constrained 2′,4′-(N-methoxy)aminomethylene and 2′,4′-aminooxymethylene and 2′-O,4′-C-aminomethylene bridged nucleoside analogues show improved potency in animal models. J Med Chem. 53 (4), 1636-1650 (2010).
- Yamamoto, T., Nakatani, M., Narukawa, K., Obika, S. Antisense drug discovery and development. Future Med Chem. 3 (3), 339-365 (2011).
- Itoh, M., Nakaura, M., Imanishi, T., Obika, S. Target gene knockdown by 2′,4′-BNA/LNA antisense oligonucleotides in zebrafish. Nucleic Acid Ther. 24 (3), 186-191 (2014).
- Han, P., et al. Hydrogen peroxide primes heart regeneration with a derepression mechanism. Cell Res. 24 (9), 1091-1107 (2014).
- Jopling, C., et al. Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature. 464 (7288), 606-609 (2010).
- Lepilina, A., et al. A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell. 127 (3), 607-619 (2006).
- McManus, M. T., Sharp, P. A. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet. 3 (10), 737-747 (2002).
- de Fougerolles, A., Vornlocher, H. P., Maraganore, J., Lieberman, J. Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov. 6 (6), 443-453 (2007).
- Kim, D. H., Rossi, J. J. Strategies for silencing human disease using RNA interference. Nat Rev Genet. 8 (3), 173-184 (2007).
- McCaffrey, A. P., et al. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol. 21 (6), 639-644 (2003).
- Raoul, C., et al. Lentiviral-mediated silencing of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS. Nat Med. 11 (4), 423-428 (2005).
- Hu-Lieskovan, S., Heidel, J. D., Bartlett, D. W., Davis, M. E., Triche, T. J. Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing’s sarcoma. Cancer Res. 65 (19), 8984-8992 (2005).
- Schiffelers, R. M., et al. Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Res. 32 (19), 149 (2004).
- Yang, X. Z., et al. Systemic delivery of siRNA with cationic lipid assisted PEG-PLA nanoparticles for cancer therapy. J Control Release. 156 (2), 203-211 (2011).
- Ko, Y. T., Hartner, W. C., Kale, A., Torchilin, V. P. Gene delivery into ischemic myocardium by double-targeted lipoplexes with anti-myosin antibody and TAT peptide. Gene Ther. 16 (1), 52-59 (2009).
- Liu, J., et al. Functionalized dendrimer-based delivery of angiotensin type 1 receptor siRNA for preserving cardiac function following infarction. Biomaterials. 34 (14), 3729-3736 (2013).
- Nam, H. Y., Kim, J., Kim, S. W., Bull, D. A. Cell targeting peptide conjugation to siRNA polyplexes for effective gene silencing in cardiomyocytes. Mol Pharm. 9 (5), 1302-1309 (2012).
- Nam, H. Y., McGinn, A., Kim, P. H., Kim, S. W., Bull, D. A. Primary cardiomyocyte-targeted bioreducible polymer for efficient gene delivery to the myocardium. Biomaterials. 31 (31), 8081-8087 (2010).
- Won, Y. W., McGinn, A. N., Lee, M., Bull, D. A., Kim, S. W. Targeted gene delivery to ischemic myocardium by homing peptide-guided polymeric carrier. Mol Pharm. 10 (1), 378-385 (2013).
- Diao, J., et al. PEG-PLA nanoparticles facilitate siRNA knockdown in adult zebrafish heart. Dev Biol. 406 (2), 196-202 (2015).
- Xiao, C., et al. Chromatin-remodelling factor Brg1 regulates myocardial proliferation and regeneration in zebrafish. Nat Commun. 7, 13787 (2016).
- Wang, F., et al. A Neutralized Noncharged Polyethylenimine-Based System for Efficient Delivery of siRNA into Heart without Toxicity. ACS Appl Mater Interfaces. 8 (49), 33529-33538 (2016).
- Kikuchi, K., et al. Retinoic acid production by endocardium and epicardium is an injury response essential for zebrafish heart regeneration. Dev Cell. 20 (3), 397-404 (2011).
- Hoshijima, K., Jurynec, M. J., Grunwald, D. J. Precise Editing of the Zebrafish Genome Made Simple and Efficient. Dev Cell. 36 (6), 654-667 (2016).
- Zu, Y., et al. TALEN-mediated precise genome modification by homologous recombination in zebrafish. Nat Methods. 10 (4), 329-331 (2013).
- Kesharwani, P., Gajbhiye, V., Jain, N. K. A review of nanocarriers for the delivery of small interfering RNA. Biomaterials. 33 (29), 7138-7150 (2012).
- Luong, D., et al. PEGylated PAMAM dendrimers: Enhancing efficacy and mitigating toxicity for effective anticancer drug and gene delivery. Acta Biomater. 43, 14-29 (2016).
- Luo, K., He, B., Wu, Y., Shen, Y., Gu, Z. Functional and biodegradable dendritic macromolecules with controlled architectures as nontoxic and efficient nanoscale gene vectors. Biotechnol Adv. 32 (4), 818-830 (2014).
- Shcharbin, D., Shakhbazau, A., Bryszewska, M. Poly(amidoamine) dendrimer complexes as a platform for gene delivery. Expert Opin Drug Deliv. 10 (12), 1687-1698 (2013).
Cite This Article
Xiao, C., Wang, F., Hou, J., Zhu, X., Luo, Y., Xiong, J. Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart. J. Vis. Exp. (137), e58054, doi:10.3791/58054 (2018).