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Neural species specific gene transfer by adenovirus vector.

通过腺病毒载体进行神经物种特异性基因转移。

基本信息

批准号：
12558087
负责人：
KIYAMA Hiroshi
金额：
$ 8.45万
依托单位：
OSAKA CITY UNIVERSITY (2001)Asahikawa Medical College (2000)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12558087/
关键词：
adenovrus Cre-loxP Promoter NRSE neuron specific Gene transfer

项目摘要

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.

我们已经尝试建立腺病毒介导的基因转移系统，使神经物种特异性表达。为此，采用了Cre-loxP系统。对神经元、星形胶质细胞或雪旺细胞特异性启动子进行体外检测。对于神经元特异性表达，使用SCG10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的特异性。星形胶质细胞表达选择GFAP启动子。这些启动子用于表达Cre，并构建了编码强通用启动子下游loxP-Stuffer-polyA-loxP-EGFP序列的腺病毒载体。通过这两种腺病毒的联合感染，尝试了神经细胞的种特异性表达。我们构建的神经元和星形胶质细胞启动子效果良好，在脑源性原代培养中表现出很高的选择性。接下来我们检测了其在大鼠脑中的特异性表达。腺病毒载体的组合被注射到大脑的不同区域。神经元特异性启动子的选择性和表达水平均较好，但各注射区表达水平差异不大。这种差异可能是由于SCG10的核心启动子；但各区域的选择性较好。对于星形胶质细胞特异性表达，无论是选择性还是表达水平都比较好，但也有少数神经元细胞表达EGFP。而在神经细胞中表达水平较低。我们还没有获得雪旺细胞特异性启动子，研究还在进行中。