一种简便、有效的心肌注射小鼠心脏特异基因操作方法--体内
Summary
在这里, 我们提出了一个小鼠心脏特异基因操作的协议。在麻醉下, 小鼠心脏通过第四肋间空间被外部化。随后, 将特定基因的腺病毒注射进心肌, 然后通过体内成像和西方印迹分析对蛋白质表达进行测量。
Abstract
特别是在心脏的基因操作明显事实心脏疾病 pathomechanisms 的调查和他们的治疗潜力。体内心脏特异基因的传递通常是通过系统性或局部分娩来实现的。应用重组腺相关病毒 9 (AAV9), 采用尾静脉全身注射治疗心脏基因表达简便、有效。然而, 这种方法需要较高的载体, 有效传导, 并可能导致 nontarget 器官基因转导。在这里, 我们描述了一个简单的, 有效的, 节省时间的方法, 心肌注射液的体内心脏特异基因操作的小鼠。在麻醉 (没有通气) 下, 胸部主要和次要肌肉被坦率地解剖, 并且小鼠心脏通过手工外在化快速地暴露通过小切口在第四肋间空间。随后, 腺病毒的编码荧光素酶 (卢克) 和维生素 D 受体 (vdr), 或短发夹 RNA (shRNA) 靶向 VDR, 注射了汉密尔顿注射器进入心肌。随后的体内成像表明, 荧光素酶成功地抗原心脏。此外, 西方印迹分析证实了成功的过度表达或沉默的 VDR 在小鼠心脏。一旦掌握, 这种技术可以用于基因操作, 以及注射细胞或其他材料, 如磁性在鼠标心脏。
Introduction
心脏病是全球发病率和死亡率的主要原因1,2。缺乏有效的治疗策略, 包括心肌梗死和心力衰竭等危及生命的心脏疾病, 吸引了对基础 pathomechanisms 的深入探索, 并确定了新的治疗方案3。对于这些科学探索, 心脏特异基因操作被广泛使用4,5。心脏基因操纵可以通过基因组编辑使用强大的转录激活器样效应核酸酶 (塔伦) 和聚集定期 interspaced 短回文重复 (CRISPR)/CRISPR 相关蛋白 9 (Cas9) 工具, 或通过交付异位基因材料 (例如, 携带感兴趣的蛋白质编码基因的病毒载体)6。虽然基因组编辑允许精确和时空基因组修改的活鼠, 它仍然是一个耗时和劳动密集型的做法6。或者, 由病毒载体或小干扰 RNA (siRNA) 复杂传递的心脏特异基因操作通常执行6。
病毒载体传递到成年鼠心脏是通过大致两个策略: 系统性或局部注射。系统性注射 cardiotropic 血清自动增值服务如 AAV9 是无创性的心脏特异基因操作7。然而, 这种方法需要一个相对较高的载体, 以有效的传导和基因表达, 并可能导致显着转导的 nontarget 器官, 如肌肉和肝脏的7。局部病毒注射是通过心肌注射或冠状动脉内传递7实现的。与心肌注射液相比, 冠状动脉内分娩可导致心脏内更均匀的病毒分布。然而, 这一技术的缺点是快速洗出的病毒载体的系统循环和转导的 nontarget 器官8, 以及它的要求设备的压力测量在操作过程中。相比之下, 心肌注射液能使心肌中的病毒保留更好, 以及特定部位的分娩, 但它无法均匀分布病毒载体7。对小动物来说, 冠状动脉内分娩在技术上很难执行, 而系统性 AAV9 注射和心肌注射则更常用4,5,7。虽然全身注射是容易执行, 常规心肌注射需要机械通气和开胸, 造成广泛的组织损伤, 是费时。
在本报告中, 我们描述了一种简单、节省时间和高效的心肌注射方法。注射荧光素酶和 vdr 的腺病毒, 或 shRNA 靶向 vdr, 被注入以操纵心脏基因表达。一旦掌握, 这种方法可以用于基因操作, 以及注射细胞或其他材料的老鼠心脏。
Protocol
Representative Results
Discussion
本报告展示了一种改进的方法, 心肌注射病毒载体的心脏基因操作, 这是修改了从心肌梗死的方法诱导的高等。13当前,在体内对特定基因功能的刻画最常涉及产生挖空或转基因小鼠3,14,15,16,17, 这是昂贵的、耗时的和劳力密集型的。或者, 通过系?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到国家杰出青年学者基金会 (81625002)、中国国家自然科学基金 (81470389、81270282、81601238) 的支持, 上海市学术研究领导 (18XD1402400) 项目教育统筹委员会高峰临床医学资助 (20152209), 上海中药肾康医院发展中心 (16CR3034A), 上海交通大学 (YG2013MS42), 上海交通大学医学院 (15ZH1003 和 14XJ10019),上海航海项目 (18YF1413000) 和蚌埠医学院研究生创新项目 (Byycx1722)。我们感谢乌尔博士以前在我们实验室的帮助。
Materials
| Equipments | |||
| Laminar flow sterile hood | Fengshi Animal Experimental Equipment Techonology Co., Ltd. (Soochow, China) | FS-CJ-2F | |
| Centrifuge | Thermo Scientific (Waltham, USA) | 75005282 | |
| Tissue grinding machine | Scientz Biotechnology Co., Ltd. (Ningbo, China) | Scientz-48 | |
| High temperature/high pressure sterilizer | Hirayama (Saitama, Japan) | HVE-50 | |
| Isoflurane vaporizer | Matrix (Orchard Park, USA) | VIP3000 | |
| IVIS Lumina III imaging system | PerkinElmer (Waltham, USA) | CLS136334 | |
| Precision balance | Sartorius (Göttingen, Germany) | 28091873 | |
| Instruments | |||
| Eppendorf pipette (100 µL) | Eppendorf (Westbury, USA) | 4920000059 | |
| Eppendorf pipette (10 µL) | Eppendorf (Westbury, USA) | 4920000113 | |
| Forceps | Shanghai Medical Instruments (Group) Ltd., Corp. | JD4020 | Curved tip |
| Hamilton syringe | Hamilton (Nevada, USA) | 80501 | Volume 50 μL |
| Micro-mosquito hemostat | F.S.T (Foster City, USA) | 13011-12 | Curved, tip width 1.3mm |
| Needle holder | Shanghai Medical Instruments (Group) Ltd., Corp. (Shanghai, China) | J32110 | |
| Surgical scissors | F.S.T (Foster City, USA) | 14002-12 | |
| 1-mL Syringe | WeiGao Group Medical Polymer Co.,Ltd. (ShangDong, China) | ||
| Materials and reagents | |||
| Anti-GAPDH antibody | CST (Danvers, USA) | #2118 | |
| Anti-Luciferase antibody | Abcam (Cambridge, UK) | ab187340 | |
| Anti-rabbit IgG | CST (Danvers, USA) | #7074 | |
| Anti-VDR antibody | Abcam (Cambridge, UK) | ab109234 | |
| Buprenorphine | Thermo Scientific (Waltham, USA) | PA175056 | |
| Chloralic hydras | LingFeng Chemical (ShangHai, China) | ||
| Cryogenic Vials | Thermo Scientific (Waltham, USA) | 375418 | 1.8 mL |
| Depilatory cream | Veet (Shanghai, China) | ||
| Dulbecco's phosphate buffered saline | Gibco (Grand Island, USA) | 14040133 | |
| Entoiodine | LiKang (Shanghai, China) | 310132 | |
| EP tube | Sarstedt (Newton, USA) | PCR001 | |
| Filter | Millipore (Bedford, USA) | Pore size 0.2 µm | |
| Isoflurane | Yipin Pharmaceutical Company (Hebei, China) | ||
| Luciferin | Promega (Madison, USA) | P1041 | |
| Lysis buffer for western blot | Beyotime (Shanghai, China) | P0013J | Without inhibitors |
| Ophthalmic cream | Apex Laboratories ( Melbourne, Australia)) | ||
| PBS | Gibco (Grand Island, USA) | 10010023 | |
| Protease inhibitor cocktail | Thermo Scientific (Waltham, USA) | 78438 | |
| 5-0 silk suture | Shanghai Medical Instruments (Group) Ltd., Corp. (Shanghai, China) | ||
| Steel ball | Scientz Biotechnology Co., Ltd. (Ningbo, China) | Width 1.5 mm | |
| Syringe needle | Kindly Medical Devices Co., Ltd. (Zhejiang, China) | 30 gauge | |
| Warm mat | Warmtact Electrical Heating Technology Co., Ltd. (Guangdong, China ) | NF-GNCW | |
References
- Yancy, C. W., et al. 2017 ACC/AHA/HFSA Focused Update of the 2013 ACCF/AHA Guideline for the Management of Heart Failure: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines and the Heart Failure Society of America. J Card Fail. 23 (8), 628-651 (2017).
- Pu, J., et al. Cardiomyocyte-expressed farnesoid-X-receptor is a novel apoptosis mediator and contributes to myocardial ischaemia/reperfusion injury. Eur Heart J. 34 (24), 1834-1845 (2013).
- He, B., et al. The nuclear melatonin receptor RORα is a novel endogenous defender against myocardial ischemia_reperfusion injury. J Pineal Res. (3), 313-326 (2016).
- Yao, T., et al. Vitamin D receptor activation protects against myocardial reperfusion injury through inhibition of apoptosis and modulation of autophagy. Antioxid Redox Signal. 22 (8), 633-650 (2015).
- He, Q., et al. Activation of liver-X-receptor alpha but not liver-X-receptor beta protects against myocardial ischemia/reperfusion injury. Circ Heart Fail. 7 (6), 1032-1041 (2014).
- Ding, J., et al. Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts. J Vis Exp. (118), (2016).
- Bish, L. T., Sweeney, H. L., Muller, O. J., Bekeredjian, R. Adeno-associated virus vector delivery to the heart. Methods Mol Biol. 807, 219-237 (2011).
- Michael, J., et al. Cardiac gene delivery with cardiopulmonary bypass. Circulation. 104 (2), 131-133 (2001).
- Lei, S., et al. Increased Hepatic Fatty Acids Uptake and Oxidation by LRPPRC-Driven Oxidative Phosphorylation Reduces Blood Lipid Levels. Front Physiol. 7, 270 (2016).
- Zhang, H. B., et al. Maintenance of the contractile phenotype in corpus cavernosum smooth muscle cells by Myocardin gene therapy ameliorates erectile dysfunction in bilateral cavernous nerve injury rats. Andrology. 5 (4), 798-806 (2017).
- Virag, J. A., Lust, R. M. Coronary artery ligation and intramyocardial injection in a murine model of infarction. J Vis Exp. (52), (2011).
- Mahmood, T., Yang, P. C. Western blot: technique, theory, and trouble shooting. N Am J Med Sci. 4 (9), 429-434 (2012).
- Gao, E., et al. A novel and efficient model of coronary artery ligation and myocardial infarction in the mouse. Circ Res. 107 (12), 1445-1453 (2010).
- Zhao, Y., et al. Novel protective role of nuclear melatonin receptor RORα in diabetic cardiomyopathy. J Pineal Res. 62 (3), (2017).
- Nduhirabandi, F., Lamont, K., Albertyn, Z., Opie, L. H., Lecour, S. Role of toll-like receptor 4 in melatonin-induced cardioprotection. J Pineal Res. 60 (1), 39-47 (2016).
- Wu, H. M., et al. JNK-TLR9 signal pathway mediates allergic airway inflammation through suppressing melatonin biosynthesis. J Pineal Res. 60 (4), 415-423 (2016).
- de Luxan-Delgado, B., et al. Melatonin reduces endoplasmic reticulum stress and autophagy in liver of leptin-deficient mice. J Pineal Res. (1), 108-123 (2016).
- Scofield, S. L., Singh, K. Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography. J Vis Exp. (117), (2016).
- Cai, B., et al. Long noncoding RNA H19 mediates melatonin inhibition of premature senescence of c-kit(+) cardiac progenitor cells by promoting miR-675. J Pineal Res. 61 (1), (2016).
- Chua, S., et al. The cardioprotective effect of melatonin and exendin-4 treatment in a rat model of cardiorenal syndrome. J Pineal Res. 61 (4), 438-456 (2016).
- Pei, H. F., et al. Melatonin attenuates postmyocardial infarction injury via increasing Tom70 expression. J Pineal Res. 62 (1), (2017).
- Yu, L., et al. Membrane receptor-dependent Notch1_Hes1 activation by melatonin protects against myocardial ischemia-reperfusion injury_ in vivo and in vitro studies. J Pineal Res. 59 (4), 420-433 (2015).
- Yu, L., et al. Melatonin rescues cardiac thioredoxin system during ischemia-reperfusion injury in acute hyperglycemic state by restoring Notch1/Hes1/Akt signaling in a membrane receptor-dependent manner. J Pineal Res. 62 (1), (2017).
- Poggioli, T., Sarathchandra, P., Rosenthal, N., Santini, M. P. Intramyocardial cell delivery: observations in murine hearts. J Vis Exp. (83), e851064 (2014).
