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.
