Neural species specific gene transfer by adenovirus vector.

我们试图建立腺病毒介导的基因转移系统，使神经种特异性表达。为此目的，采用Cre-loxP系统。在体外检查神经元、星形胶质细胞或雪旺细胞特异性启动子。对于神经元特异性表达，使用SCG 10的基本启动子，并附加神经元特异性沉默元件（NRSE）。这种修饰显著增加了神经元特异性。对于星形胶质细胞表达，选择GFAP启动子。这些启动子用于Cre的表达，并构建了另外的腺病毒载体，其编码强通用启动子下游的loxP-Stuffer-polyA-loxP-EGFP序列。通过这两种腺病毒的共感染，尝试神经种特异性表达。我们构建的用于神经元和星形胶质细胞的启动子工作良好，并且在培养物如脑源性原代培养物中显示出非常高的选择性。接下来，我们检查了大鼠脑中表达的特异性。将腺病毒载体的组合注射到不同的脑区域中。神经元特异性启动子的选择性和表达水平良好，然而观察到注射区域之间表达水平的微小差异。这种差异可能是由于SCG 10的核心启动子;然而，在各个区域中的选择性良好。对于星形胶质细胞特异性表达，选择性和表达水平均相对较好，但也有少量神经元细胞表达EGFP，但神经元细胞表达水平较低。目前尚未获得雪旺细胞特异性启动子，相关研究仍在进行中。

We have attempted to establish the adenovirus-mediated gene-transfer system, which enables neural species-specific expression. For this aim, Cre-loxP system was employed. Neuron, astrocyte, or Schwann cell specific promoters were examined in vitro. For the neuron specific expression, the basic promoter for SCG10 was used, and additional neuron specific silencer elements (NRSE) were attached. This modification increased the neuron specificity significantly. For the astrocyte expression, GFAP promoter was selected. Those promoters were used for the expression of Cre, and additional adenovirus vector, which encodes loxP-Stuffer-polyA-loxP-EGFP sequence downstream of a strong general promoter, was constructed. By the co-infection of these two adenoviruses, neural specie specific expression was attempted. The promoters we constructed for neurons and astrocytes worked well, and showed very high selectivity in culture such as the brain derived primary culture. Next we examined the specificity of the expression in rat brain. The combination of the adenovirus vectors was injected into various brain regions. The selectivity and expression level of the neuron specific promoter was nice, however the minor differences in the expression level among the injected regions were observed. This difference may be due to the core promoter of SCG10 ; nevertheless the selectivity in various regions was good. For the astrocyte specific expression, both selectivity and expression level was relatively good, but a few neuronal cells expressing EGFP was also found. However the expression level in neuronal cells were low. We have not obtained the Schwann cell specific promoter yet, and the study is on going.