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Spatiotemporal knockout by an Adenovirus-Targeting System.

通过腺病毒靶向系统进行时空敲除。

基本信息

批准号：
10044227
负责人：
KIYAMA Hiroshi
金额：
$ 8.32万
依托单位：
Asahikawa Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10044227/
关键词：
injured neuron adenovirus neuron-specific SCGIO Cre recombinase GAP-43 nestin c-jun LacZ トランスジェニックマウス atf-3 サイレントノックアウト c-Jun 神経再生 Cre LoxP 神経細胞損傷遺伝子相同組換え

项目摘要

The current experiment can be dissected down into two principles. First, it is essential for the recombinational events to occur only in the cells in question (ie, injured neurons in this case). Second, it is of large benefit to restrict the number of gene manipulation before observing the outcome. When a non-restrictive promoter is used to drive the expression from the adenoviruses, we were unable to target the Cre expression solely to the axonally-damaged neurons but would widely rearrange the neighboring cells. If the gene in question is closely related to the survival and/or maintenance of a cell, it is crucial to restrict the expression in a strict temporal and spatial manner. We then tried to gain some neuronal specificity within the adenovirus' promoter. As the size matters when recombining into the expression module of an adenovirus, we initially chose the relatively small SCG10/REST system. This has led to a relatively neuronal restrictive expression of a reporter gene. Howeve … More r, when tested with a Cre expressing system, the switch of the expressivity wasn't sharp enough to restrict the recombination to the damaged neurons. We next rested transgenic mice with a pan-neuronal specific promoter such as GAP-43 or nestin enhancer. These in turn showed some problems of their own. Nevertheless, it is of utmost importance to strictly regulate the gene expression and we hope to realize this through some refinements of these. We additionally obtained some interesting insights of the role of the target genes during nerve injury. c-Jun (especially its phosphorylation) is believed to play a part in the decision of cell fate during neuronal cell death. We do agree on this principle and therefore chose the molecule for the target of our silent-knockout (=the background mice for the conditional gene targeting). In an independent experiment, we showed that an additional member of the AP-1 family, Atf-3 might be as much crucial and cooperate with c-jun during death/survival decision. We are now Flaming to apply the current experiment paradigm for this molecule as well. Less

目前的实验可以分解为两个原则。首先，重组事件必须只发生在有问题的细胞中(即，在这种情况下，受损的神经元)。其次，在观察结果之前限制基因操作的次数是很有好处的。当使用非限制性启动子驱动腺病毒的表达时，我们不能仅针对轴突损伤的神经元表达Cre，而是广泛地重排邻近细胞。如果所讨论的基因与细胞的生存和/或维持密切相关，那么以严格的时间和空间方式限制表达是至关重要的。然后，我们试图在腺病毒的启动子中获得一些神经元特异性。由于重组腺病毒表达模块时大小很重要，我们最初选择了相对较小的SCG10/REST系统。这导致了报告基因在神经元上的相对限制性表达。然而，…更重要的是，当用Cre表达系统测试时，表达能力的变化不够尖锐，不足以将重组限制在受损的神经元上。接下来，我们让带有泛神经元特异性启动子的转基因小鼠休息，比如GAP-43或Nestin增强子。这些反过来又显示出它们自己的一些问题。然而，严格调控基因表达是最重要的，我们希望通过一些改进来实现这一点。此外，我们还对靶基因在神经损伤中的作用获得了一些有趣的见解。C-jun(尤其是其磷酸化)被认为在神经细胞死亡过程中对细胞命运起着决定作用。我们确实同意这一原则，因此选择了分子作为我们沉默基因敲除的目标(=有条件基因打靶的背景鼠)。在一项独立的实验中，我们证明了AP-1家族的另一个成员ATF-3可能同样至关重要，并在死亡/生存决定中与c-Jun合作。我们现在正在努力将目前的实验范式也应用于这种分子。较少