CORE--VECTOR DESIGN AND PRODUCTION

载体设计和生产核心将提供重组 基于不能复制的单纯疱疹病毒（HSV）的载体， 在原代培养的神经元中转移和表达选定的转基因 （项目2）和海马神经元缺血前和缺血期间， vivo（项目3）。该策略将用于直接测试 选择已知的和新的基因，通过它们的区域表达来鉴定， 缺血性脑（项目1），在缺血性神经元死亡的调节。 基于HSV的载体将通过同源重组一个 含有在人的控制下的选择的基因的质粒 巨细胞病毒立即早期启动子（HCMV-IEp）和lacZ报告基因 基因插入ICP 4复制的胸苷激酶（tk）基因座， 在互补E5细胞中繁殖的无缺陷HSV载体。的 转基因RNA的体外表达将通过北方印迹证实， Western blot检测蛋白在体外的表达。梯度纯化的 然后将载体原种用于转移和表达转基因 产品体外（项目2）和体内（项目3）。

The vector Design and Production Core will provide recombinant replication-incompetent herpes simplex virus (HSV)-based vectors to transfer and express selected transgenes in neurons in primary culture (Project 2) and in hippocampal neurons prior to and during ischemia in vivo (Project 3). This strategy will be used to directly test the role of selected known and novel genes, identified by their regional expression in ischemic brain (Project 1), in the regulation of ischemic neuronal death. HSV-based vectors will be constructed by homologous recombination of a plasmid-containing the selected gene under the control of the human cytomegalovirus immediate-early promoter (HCMV-IEp) and the lacZ reporter gene-into the thymidine kinase (tk) locus of an ICP4-replication- incompetent HSV vector propagated in complementing E5 cells. The expression of transgene RNA in vitro will be confirmed by Northern blot, and expression of the protein in vitro by Western blot. Gradient purified vector stocks will then be used to transfer and express the transgene product in vitro (Project 2) and in vivo (Project 3).