Ding Jian, Lin Zhi-Qiang, Jiang Jian-Ming, Seidman Christine E, Seidman Jonathan G, Pu William T, Wang Da-Zhi
Department of Cardiology, Boston Children's Hospital; Department of Pediatrics, Harvard Medical School;
Department of Cardiology, Boston Children's Hospital; Department of Pediatrics, Harvard Medical School.
J Vis Exp. 2016 Dec 17(118):54787. doi: 10.3791/54787.
Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.
通过在小鼠模型中经心肌递送遗传物质来控制特定基因的表达或活性，有助于研究基因功能。还可以确定它们在心脏中的治疗潜力。对小鼠心脏进行体内分子干预的方法有限。基于重组腺相关病毒（rAAV）的基因组工程已被用作体内心脏基因操作的重要工具。该技术的具体优势包括高效、高特异性、低基因组整合率、最小免疫原性和最小致病性。在此，描述了构建、包装和纯化rAAV9载体的详细步骤。将rAAV9皮下注射到新生幼崽中，可在小鼠心脏中实现目的基因的强表达或有效敲低，而在肝脏和其他组织中则不会。使用心脏特异性TnnT2启动子，可在心脏中获得GFP基因的高表达。此外，当使用rAAV9-U6-shRNA时，心脏中的靶mRNA受到抑制。掌握rAAV9技术的实用知识可能对心血管研究有用。
