Spatiotemporal knockout by an Adenovirus-Targeting System.

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非常有益的。当使用非限制性启动子来驱动腺病毒的表达时，我们不能将Cre的表达仅靶向轴突损伤的神经元，而是会广泛地重新排列邻近细胞。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其表达是至关重要的。然后，我们试图在腺病毒启动子中获得一些神经元特异性。由于重组成腺病毒表达模块时，大小很重要，我们最初选择了相对较小的SCG10/REST系统。这导致了报告基因的相对神经元限制性表达。然而，当使用Cre表达系统进行测试时，表达性的转换不够灵敏，无法将重组限制在受损神经元上。接下来，我们用泛神经元特异性启动子（如GAP-43或巢蛋白增强子）休息转基因小鼠。这些因素反过来也显示出它们自身的一些问题。然而，严格调控基因表达是至关重要的，我们希望通过对这些基因的一些完善来实现这一点。我们还对靶基因在神经损伤过程中的作用获得了一些有趣的见解。c-Jun（特别是其磷酸化）被认为在神经元细胞死亡过程中决定细胞命运。我们确实同意这一原则，因此选择了分子作为我们沉默敲除的目标（=条件基因靶向的背景小鼠）。在一项独立实验中，我们发现AP-1家族的另一个成员Atf-3可能同样重要，并在死亡/生存决策中与c-jun合作。我们现在正在对这个分子应用当前的实验范例。少

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