REGULATABLE INHIBITION OF ANY GENE IN TRANSGENIC MICE

了解哺乳动物基因的功能需要控制它们在动物体内表达的方法。敲除小鼠提供了许多基因的重要信息，但缺乏分级控制和基因改变的不可逆性限制了它们的用途，特别是对于在发育中起重要作用的基因。本提案描述了一种创建转基因小鼠的方法，其中使用四环素（tet）调控启动子驱动的RNA Padlock抑制剂，可以以组织特异性，可逆和定量的方式抑制任何所需的基因。RNA挂锁通过在反义RNA和目标mRNA之间形成拓扑连接来灭活特定基因的表达。在对其进行研究的系统中，RNA Padlocks已被证明在抑制特定基因表达方面比传统的反义方法更有效。原理证明将通过创建一个针对NMDA受体（nr - 1）亚基1的挂锁来提供，NMDA受体与学习和神经元可塑性有关。然后构建在tet控制下表达该抑制剂的载体，并测试其在神经细胞系中抑制nr - 1表达的能力。在证明tet可调节抑制作用之后，将创建转基因小鼠，其中nr - 1在给药四环素衍生物后在海马中被特异性抑制。建议的商业应用：基因敲除小鼠目前是了解基因功能的最佳工具，但对许多基因敲除是致命的或阻止正常发育。因此，需要一种制造条件敲除的方法，从而可以在所需的时间和特定组织中关闭和打开基因。这种方法对于研究目的和药物开发中的靶点验证非常有用。通过基因转移方法引入有效的、可调节的基因抑制剂的能力也是某些类型基因治疗的关键，其目的是在疾病发作时根据需要控制天然基因，从而最大限度地减少不良影响。

Understanding the function of mammalian genes requires methods for controlling their expression in the animal. Knockout mice provide important information about many genes, but the lack of graded control and irreversibility of the genetic alteration limits their usefulness, particularly for genes that play important roles in development. This proposal describes a method for creating transgenic mice in which any desired gene can be inhibited in a tissue-specific, reversible, and quantitative manner, using RNA Padlock inhibitors driven by tetracycline (tet)-regulated promoters. RNA Padlocks inactivate the expression of specific genes by forming a topological linkage between an antisense RNA and the target mRNA. In systems where they have been studied, RNA Padlocks have been demonstrated to be much more effective at inhibiting the expression of specific genes than conventional antisense approaches. Proof of principle will be provided by creating a Padlock directed against subunit l of the NMDA receptor (NR-l), which is implicated in learning and neuronal plasticity. A vector expressing this inhibitor under tet control will then be constructed and tested for its ability to inhibit NR-l expression in a neuronal cell line. Demonstration of tet-regulatable inhibition will be followed by the creation of transgenic mice in which NR-l is inhibited specifically in the hippocampus upon administration of a tetracycline derivative. PROPOSED COMMERCIAL APPLICATIONS: Knockout mice are currently the best tool for understanding gene function, yet for many genes knockouts are lethal or prevent normal development. There is thus a need for a method for making conditional knockouts, whereby the gene can be turned off and on at desired times and in specific tissues. Such a method would be invaluable for research purposes and very useful for target validation in drug development. The ability to introduce potent, regulatable gene inhibitors by gene transfer methods is also key to some types of gene therapy, where the aim is to control of a natural gene as needed, at times of a disease flare-up, thus minimizing adverse effects.