A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice
Summary
Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.
Abstract
Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.
Introduction
Cardiac disease is the leading cause of morbidity and mortality worldwide1,2. The lack of effective therapeutic strategies for life-threatening heart conditions including myocardial infarction and heart failure attracts intensive exploration of underlying pathomechanisms and identification of novel therapeutic options3. For these scientific explorations, cardiac-specific gene manipulation is widely used4,5. Cardiac gene manipulation can be achieved by genome editing using the powerful transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) tools, or by delivery of ectopic genetic materials (e.g., virus vectors carrying genes encoding proteins of interest)6. Though genome editing allows precise and spatiotemporal genome modifications in living mice, it is still a time-consuming and labor-intensive practice6. Alternatively, cardiac-specific gene manipulation by virus vector or small interfering RNA (siRNA) complex delivery are routinely performed6.
Virus vector delivery to the adult mouse heart is achieved by roughly two strategies: systemic or local injection. Systemic injection of cardiotropic serotype of AAVs such as AAV9 is noninvasive for cardiac specific gene manipulation7. However, this method requires a relatively high amount of vector necessary for efficient transduction and gene expression, and may result in significant transduction of nontarget organs such as the muscle and liver7. Local virus injection is achieved by intramyocardial injection or intracoronary delivery7. Intracoronary delivery leads to a more even distribution of virus within the heart compared to intramyocardial injection. However, the disadvantages of this technique are the rapid wash out of viral vectors to the systemic circulation and transduction in nontarget organs8, and its requirement of devices for pressure measurement during the operation. By contrast, intramyocardial injection enables better virus retention in the myocardium as well as site specific delivery, but it fails to evenly distribute viral vector7. For small animals, intracoronary delivery is technically difficult to perform, while systemic AAV9 injection and intramyocardial injection are more commonly practiced4,5,7. Though systemic injection is easy to perform, conventional intramyocardial injection requires mechanical ventilation and thoracotomy, causes extensive tissue damage, and is time-consuming.
In this report, we described an easy, time-saving, and highly efficient method for intramyocardial injection. Adenovirus encoding luciferase and VDR, or shRNA targeting VDR, was injected to manipulate cardiac gene expression. Once mastered, this method can be used for gene manipulation, as well as injection of cells or other materials in the mouse heart.
Protocol
Representative Results
Discussion
The current report demonstrates a modified technique for intramyocardial injection of viral vectors for cardiac gene manipulation, which was modified from a method for myocardial infarction induction by Gao et al.13 Currently, in vivo characterization of specific gene functions most often involve the generation of knockout or transgenic mice3,14,15,16<s…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by National Science Fund for Distinguished Young Scholars (81625002), National Natural Science Foundation of China (81470389, 81270282, 81601238), Program of Shanghai Academic Research Leader (18XD1402400), Shanghai Municipal Education Commission Gaofeng Clinical Medicine Grant Support (20152209), Shanghai Shenkang Hospital Development Center (16CR3034A), Shanghai Jiao Tong University (YG2013MS42), Shanghai Jiao Tong University School of Medicine (15ZH1003 and 14XJ10019), Shanghai Sailing Program (18YF1413000), and Postgraduate Innovation Program of Bengbu Medical College (Byycx1722). We thank Dr. Erhe Gao for his previous help in our lab.
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 | |
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