Spatial memory performance declines in both normal aging and Alzheimer's disease. This cognitive deficit is related to hippocampus dysfunction. Gene therapy using neurotrophic factors like Glial cell line-derived neurotrophic factor (GDNF) emerges as a promising approach to ameliorate age-related cognitive deficits. We constructed a two vector regulatable system (2VRS) which consists of a recombinant adenoviral vector (RAd) harboring a Tet-Off bidirectional promoter flanked by GDNF and Green Fluorescent Protein (GFP) genes. A second adenovector, RAd-tTA, constitutively expresses the regulatory protein tTA. When cells are cotransduced by the 2VRS, tTA activates the bidirectional promoter and both transgenes are expressed. In the presence of the antibiotic doxycycline (DOX) transgene expression is silenced. We tested the 2VRS in CHO-K1 cells where we observed a dose-dependent GFP expression that was completely inhibited by DOX (1 mg/ml). The 2VRS injected in the hippocampal CA1 region transduced both neurons and astrocytes and was efficiently inhibited by DOX added to the drinking water. In order to assess GDNF biological activity we injected 2VRS and its Control (CTRL) vector in the hypothalamus and monitored body weight for one month. The results showed that GDNF retards weight recovery 6 days more than CTRL. In conclusion, our 2VRS demonstrated optimal GFP expression and showed a bioactive effect of transgenic GDNF in the brain.

中文翻译：

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用于大鼠海马中 GDNF 和 GFP 递送的可调节腺载体系统

空间记忆能力在正常衰老和阿尔茨海默病中都会下降。这种认知缺陷与海马体功能障碍有关。使用神经营养因子（如神经胶质细胞系衍生的神经营养因子（GDNF））的基因治疗成为改善与年龄相关的认知缺陷的有前途的方法。我们构建了一个双载体调节系统 (2VRS)，它由一个重组腺病毒载体 (RAd) 组成，该载体带有一个 Tet-Off 双向启动子，两侧是 GDNF 和绿色荧光蛋白 (GFP) 基因。第二个腺载体 RAd-tTA 组成型表达调节蛋白 tTA。当细胞被 2VRS 共转导时，tTA 会激活双向启动子并表达两种转基因。在抗生素强力霉素 (DOX) 存在下，转基因表达被沉默。我们在 CHO-K1 细胞中测试了 2VRS，我们观察到剂量依赖性 GFP 表达被 DOX (1 mg/ml) 完全抑制。注射到海马 CA1 区的 2VRS 转导神经元和星形胶质细胞，并被添加到饮用水中的 DOX 有效抑制。为了评估 GDNF 生物活性，我们在下丘脑注射 2VRS 及其对照 (CTRL) 载体并监测体重一个月。结果表明GDNF比CTRL多延迟6天的体重恢复。总之，我们的 2VRS 显示出最佳的 GFP 表达，并显示出转基因 GDNF 在大脑中的生物活性作用。注射到海马 CA1 区的 2VRS 转导神经元和星形胶质细胞，并被添加到饮用水中的 DOX 有效抑制。为了评估 GDNF 生物活性，我们在下丘脑注射 2VRS 及其对照 (CTRL) 载体并监测体重一个月。结果表明GDNF比CTRL多延迟6天的体重恢复。总之，我们的 2VRS 显示出最佳的 GFP 表达，并显示出转基因 GDNF 在大脑中的生物活性作用。注射到海马 CA1 区的 2VRS 转导神经元和星形胶质细胞，并被添加到饮用水中的 DOX 有效抑制。为了评估 GDNF 生物活性，我们在下丘脑注射 2VRS 及其对照 (CTRL) 载体并监测体重一个月。结果表明GDNF比CTRL多延迟6天的体重恢复。总之，我们的 2VRS 显示出最佳的 GFP 表达，并显示出转基因 GDNF 在大脑中的生物活性作用。