CONSTRUCTION OF ADENOVIRUS VECTORS

腺病毒载体介导的基因转移特别吸引人 对于对标准的转染法无效的细胞， 例如磷酸钙沉淀、脂质体或 电穿孔。这包括原代细胞培养和其他 高度分化的细胞，包括未分裂的细胞。对于 同样的原因，腺病毒载体是治疗IN的首选方法 原位基因转移。此外，腺病毒载体可用于 在一个细胞中同时表达几个外源基因，或者 以规定的时间间隔。基因表达水平是可以控制的 根据使用的病毒剂量。这个项目的目的是 构建体外使用的复制缺陷载体。这些 载体将指导广泛的蛋白质的表达，这些蛋白质 是NIDR Intral部门的调查人员感兴趣的 研究。这些向量将被用于研究信号转导， 细胞外基质与唾液腺生理学。具体地说， 该合同的第一个目标将是培养和纯化质粒 包含腺病毒序列。第二个将是 重组质粒来生成腺病毒载体。第三个目标 该项目的主要任务是扩增分离的腺病毒斑块，并 把它们提纯成病毒库。这些试剂将是独一无二的 不能从商业渠道获得。

Adenoviral vector-mediated gene transfer is particularly attractive for cells that are refractory to standard methods of transfection, such as calcium phosphate precipitation, liposomes or electroporation. This includes primary cell cultures and other highly differentiated cells, including non-dividing cells. For the same reasons, adenoviral vectors are the method of choice for in situ gene transfer. In addition, adenoviral vectors may be used to express several exogenous genes in a cell, either simultaneously or at defined intervals. Levels of gene expression can be controlled by the dose of virus used. The purpose of this project is to construct replication-deficient vectors for in vitro use. These vectors will direct the expression of a wide range of proteins that are of interest to investigators in the NIDR Division of Intramural Research. The vectors will be used to study signal transduction, extracellular matrix and salivary gland physiology. Specifically, the first goal of the contract will be to grow and purify plasmids containing adenovirus sequences. The second will be to recombine plasmids to generate adenovirus vectors. The third goal of the project will be to amplify isolated adenovirus plaques and to purify them into virus stocks. These reagents will be unique and not available from commercial sources.