RIBOZYME MEDIATED KNOCKOUT OF THE CYSTATIN S GENE

核酶介导的胱抑素 S 基因敲除

• 批准号: 2132565
• 负责人: PHYLLIS Ann SHAW
• 金额: $ 4.11万
• 依托单位: MOUNT SINAI SCHOOL OF MEDICINE OF CUNY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2132565
• 关键词: developmental genetics; gene deletion mutation; genetically modified animals; growth /development; laboratory rat; protease inhibitor; ribozymes; submandibular gland; transfection /expression vector

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.

胱抑素超家族的成员是半胱氨酸蛋白酶抑制剂 包括细胞内的stefins，分泌的半胱氨酸蛋白酶抑制剂， 高分子量激肽原。 虽然所有这些蛋白酶 抑制剂在体外抑制半胱氨酸蛋白酶，它们在体内的功能 还没有被明确地证明。 因为我们的长期目标 是了解胱抑素在生长，分化和 唾液腺的形态发生，我们建议检查生理 半胱氨酸蛋白酶抑制剂S（CysS）的功能，其在下颌下 大鼠的腺体 CysS基因特别适合于 审查其功能，因为它是在特定阶段表达的 (28天）的大鼠下颌下腺的出生后发育， 而在成年动物中则是关闭的。 有待检验的假设是， CysS是大鼠下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 为了研究CysS基因的敲除如何影响 “正常”大鼠下颌下腺发育。 核酶是催化性RNA 在体外和体内切割特异性RNA靶序列的分子， 导致靶基因表达的不同水平的降低， 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中尝试，但是， 在这个建议中所概述的，它有很好的机会实现。 没有CysS的大鼠是否会有强烈的或一些微妙的差异， 下颌下腺的发育与正常相比 将检查同窝仔。 这些实验将使今后的研究 为了确定核酶靶向敲除或抑制 其他唾液基因，以确定它们的功能。 事实上，类似的策略可以用于敲除任何其他基因。 哺乳动物系统来研究其功能。 具体目的是：1）构建适合于人的CysS核酶载体 进行CysS基因敲除实验，并检测其表达，催化 体外对大鼠CysS mRNA的活性。 2)微量注射CysS 将核酶构建体插入单细胞卵的雄性原核中以产生 转基因老鼠 3)识别携带核酶CysS的动物 转基因，并检查转基因对下颌下 CysS基因敲除动物的腺体发育。