Wacker Bradley K, Bi Lianxiang, Dichek David A
Department of Medicine, University of Washington.
Department of Medicine, University of Washington;
J Vis Exp. 2018 May 6(135):56982. doi: 10.3791/56982.
The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.
该方法的目标是将转基因导入兔双侧颈总动脉分离段的内皮中。该方法实现了局部内皮选择性转基因，从而使研究人员能够确定内皮表达的转基因的生物学作用，并量化大动脉内皮细胞中DNA序列的体内转录活性。该方法采用手术分离兔颈总动脉，并通过动脉切开术将表达转基因的病毒载体导入动脉腔。载体在腔内短暂孵育，随后吸出腔内内容物，足以在内皮中实现转基因的高效持久表达，在分离的动脉段外未检测到转导或表达。该方法允许在正常动脉和人类血管疾病模型中评估转基因产物的生物学活性，同时避免因将基因传递靶向其他部位（如肝脏）或通过种系转基因将基因构建体传递到内皮的替代方法而可能引起的全身效应。该方法的应用受到以下因素的限制：需要熟练的外科医生和麻醉师、设备完善的手术室、购买和饲养兔子的成本，以及基因传递载体构建和使用方面的专业知识。用该方法获得的结果包括：转基因相关的动脉结构、细胞数量、细胞外基质或血管舒缩功能改变；动脉炎症的增加或减少；血管细胞凋亡的改变；以及内膜增生或动脉粥样硬化等疾病的进展、延缓或消退。该方法还允许测量天然和合成DNA调控序列改变内皮细胞中转基因表达的能力，提供的结果包括：转基因mRNA水平、转基因蛋白水平和转基因酶活性水平。
