RIBOZYME MEDIATED KNOCKOUT OF THE CYSTATIN S GENE

半胱氨酸蛋白酶抑制剂是半胱氨酸蛋白酶的超家族成员 这包括细胞内的Stefins，分泌的半胱氨酸，以及 高分子量激肽原。尽管所有这些蛋白酶 半胱氨酸蛋白酶抑制剂的体外抑制作用及其体内作用 还没有得到明确的证明。因为我们的长期目标是 是为了了解半胱氨酸氨基转移酶在生长、分化和 唾液腺的形态发生，我们建议检查生理 颌下腺表达的半胱氨酸氨基转移酶S的功能 老鼠的腺体。CysS基因特别适合于 检查其功能，因为它是在特定阶段表达的 (28天)大鼠颌下腺的出生后发育， 在成年动物身上是关闭的。需要检验的假设是 半胱氨酸是大鼠颌下腺发育所必需的。我们 计划培育携带抗CysS核酶的转基因大鼠 基因，以研究CysS基因敲除如何影响 “正常”大鼠颌下腺发育。核酶是一种催化的RNA 在体外和体内切割特定RNA靶标序列的分子， 导致不同程度的靶基因表达降低， 事件达到完全压制的程度。因此，核酶是有用的。 用于研究动物发育过程中的基因功能。据我们所知， 核酶介导的基因敲除还没有在大鼠身上尝试过，但作为 在这项提案中概述的它有很好的机会实现。 一只没有CysS的大鼠在 颌下腺发育与正常对照的研究 将对垃圾进行检查。这些实验将使未来的研究成为可能 确定以核酶为靶点的基因敲除或抑制 通过类似的程序确定其他唾液基因的功能(S)。 事实上，类似的策略也可以用来敲除其他基因。 研究其功能的哺乳动物系统。 具体目的是：1)构建合适的CysS核酶载体 进行CysS基因敲除实验，并检测其表达情况，并催化 体外抗大鼠CysS基因的活性。2)微量注射CysS 核酶构建到单细胞卵子雄性原核中生产 转基因大鼠。3)鉴定携带核酶CysS的动物 并检测转基因对颌下腺的影响。 CysS基因敲除动物的腺体发育。

Members of the cystatin superfamily are cysteine proteinase inhibitors that include the intracellular stefins, the secreted cystatins, and the high molecular weight kininogens. Although all these proteinase inhibitors inhibit cysteine proteinases in vitro, their in vivo functions have not been unequivocally demonstrated. Since our long-term objective is to understand the role of cystatins in growth, differentiation and morphogenesis of salivary glands, we propose to examine the physiological function of cystatin S (CysS), which is expressed in the submandibular gland of the rat. The CysS gene lends itself particularly well for examination of its function, since it is expressed at a particular stage (28 days) of postnatal development of the submandibular gland of the rat, and is turned off in adult animals. The hypothesis to be tested is that CysS is necessary for the development of the rat submandibular gland. We plan to generate transgenic rats that carry a ribozyme against the CysS gene, in order to investigate how the knockout of the CysS gene affects "normal" rat submandibular gland development. Ribozymes are catalytic RNA molecules that cleave specific RNA target sequences, in vitro and in vivo, resulting in various levels of reduced expression of the target gene, event to the extent of complete suppression. Thus, ribozymes are useful for studying gene function during animal development. To our knowledge, ribozyme-mediated gene knockout has not been attempted in the rat, but as outlined in this proposal it has a very good chance of being accomplished. Whether a rat without CysS will have a strong or some subtle difference in development of the submandibular gland in comparison to its normal littermate will be examined. These experiments will permit future studies to determine the effects of ribozyme-targeted knockout or suppression of other salivary genes by similar procedures to determine their function(s). In fact, a similar strategy can be used to knockout other genes i any mammalian system to study its function. The Specific Aims are to: 1) Construct CysS ribozyme vectors suitable for CysS gene knockout experiments, and test its expression, and catalytic activity against rat CysS mRNA in vitro. 2) Microinject the CysS ribozyme construct into male pronuclei of one-cell eggs for the production of transgenic rats. 3) Identify animals carrying the ribozyme CysS transgene, and to examine the effects of the transgene upon submandibular gland development in the animals with the knocked-out CysS gene.