CORE--VECTOR DESIGN AND PRODUCTION

载体设计和生产核心将提供重组 复制不能单纯疱疹病毒(HSV)为基础的载体 选择性转基因在原代培养神经元中的转移和表达 (方案2)和缺血前和缺血期间的海马神经元 Vivo(项目3)。这一战略将被用来直接测试 选定的已知和新基因，通过它们在 缺血性脑(项目1)，在调节缺血性神经元死亡中的作用。 基于HSV的载体将通过同源重组构建 含有人类控制的所选基因的质粒组 巨细胞病毒立即早期启动子(HCMV-IEP)和LacZ报告基因 基因-进入ICP4的胸苷激酶(Tk)基因-复制- 失活的HSV载体在互补的E5细胞中繁殖。这个 用Northern印迹法证实转基因RNA在体外的表达。 Western印迹法检测该蛋白在体外的表达。梯度提纯 然后，载体股票将被用于转移和表达转基因 体外产品(项目2)和体内产品(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).