Neuron-specific expression of therapeutic proteins:: Evaluation of different cellular promoters in recombinant adenoviral vectors
Neuron-specific expression of therapeutic proteins:: Evaluation of different cellular promoters in recombinant adenoviral vectors
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DOI：
10.1006/mcne.2000.0929
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发表时间：
2001-01-01
影响因子：
3.5
通讯作者：
Bähr, M
中科院分区：
文献类型：
--
作者：
Kügler, S;Meyn, L;Bähr, M
为了实现抗凋亡蛋白的神经元限制性表达，在腺病毒载体的背景下研究了细胞启动子的表达谱。在原代神经元与其必需饲养细胞的共培养中，突触蛋白 1 基因和微管蛋白 α1 基因启动子均具有严格的神经元特异性。神经元特异性烯醇化酶基因启动子在培养的海马神经元中仅表现出弱活性，并且在小脑颗粒细胞制备物中不具有神经元特异性。通过达到几乎 100% 的转导效率，我们能够使用分化和完全未分化的海马神经元生成“准转基因”原代神经元培养物。在功能测定中，我们使用突触蛋白启动子来评估 Bcl-X-L 过表达对钾撤除诱导的小脑颗粒神经元细胞凋亡的影响。我们发现 caspase-9 和 -3 激活和细胞凋亡几乎完全受到抑制，表明线粒体途径在这种神经元细胞死亡范例中发挥着重要作用。突触蛋白启动子作为强全神经元启动子的优异适用性通过其在成年大鼠体内各个脑区中受限的神经元活动得到进一步证明。
In order to achieve neuron-restricted expression of antiapoptotic proteins, cellular promoters were investigated for their expression profiles in the context of adenoviral vectors. Both the synapsin 1 gene and the tubulin alpha1 gene promoters were strictly neuron specific in cocultures of primary neurons with their essential feeder cells. The neuron-specific enolase gene promoter exhibited only weak activity in cultured hippocampal neurons and was not neuron specific in preparations of cerebellar granule cells. By attaining virtually 100% transduction efficiency we were able to generate "quasi-transgenic" primary neuron cultures using both differentiated and completely undifferentiated hippocampal neurons. In a functional assay, we used the synapsin promoter to evaluate the effect of Bcl-X-L, overexpression on potassium-withdrawal-induced apoptosis of cerebellar granule neurons. We found nearly complete inhibition of caspase-9 and -3 activation and apoptosis, indicating a major role for mitochondrial pathways in this paradigm of neuronal cell death. The excellent suitability of the synapsin promoter as a strong panneuronal promoter was further demonstrated by its restricted neuronal activity in various brain regions of adult rats in vivo.