Fu Yanan, Jiang Wenlong, Zhao Yichao, Huang Yuli, Zhang Heng, Wang Hongju, Pu Jun
Department of Cardiology, The First Affiliated Hospital of Bengbu Medical College; Department of Cardiology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University.
Department of Cardiology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University.
J Vis Exp. 2018 Apr 16(134):57074. doi: 10.3791/57074.
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.
特别是在心脏中进行基因操作可显著增强对心脏病发病机制及其治疗潜力的研究。体内心脏特异性基因递送通常通过全身或局部递送实现。通过尾静脉进行全身注射，利用重组腺相关病毒9（AAV9）操纵心脏基因表达既简单又高效。然而，这种方法需要相对大量的载体才能实现高效转导，并且可能导致非靶器官基因转导。在此，我们描述一种用于小鼠体内心脏特异性基因操作的简单、高效且省时的心肌内注射方法。在麻醉状态下（无需通气），钝性分离胸大肌和胸小肌，通过第四肋间间隙的小切口手动将小鼠心脏快速暴露。随后，用汉密尔顿注射器将编码荧光素酶（Luc）和维生素D受体（VDR）的腺病毒，或靶向VDR的短发夹RNA（shRNA）注射到心肌中。随后的体内成像显示荧光素酶在心脏中成功特异性过表达。此外，蛋白质印迹分析证实小鼠心脏中VDR成功过表达或沉默。一旦掌握，该技术可用于小鼠心脏中的基因操作，以及细胞或其他材料（如纳米凝胶）的注射。
