The goal of this project is to delineate the cis-acting elements which are responsible for regulation of human CFTR expression. Results from this work should enhance our understanding of the pathogenesis of CF (progression and organ specificity) and potentially contribute to the development of novel gene therapy strategies.The elements which regulate CFTR expression are currently poorly defined. In the past, plasmid expresssion vectors incorporating putative promoter elements and reporter genes have been used to study CFTR expression in human cells. However, these stuides have generally yielded disappointing results because the exptected patterns and/or level of CFTR expression have not been faithfully reproduced. One explanation is that the most important regulatory sequences have not been included in these small constructs. In order to faciliate assessment of the role of potential regulatory elements including those which may exist in distant flanking or large intronic sequences, expression vectors with larger DNA capacity would be useful. Yeast artificial chromosome (YAC) vectors are ideally suited for such studies since they can be used to manipulate and analyze large fragments of human DNA. The proposed study will utilize a YAC vector system to delineate the cis-acting regulatory elements that control human CFTR expression. We have shown that two CFTR-containing YACs (320 and 620 kb) derived from different libraries can produce both full-length CFTR mRNA and functional protein when introduced into Chinese hamster ovary (CHO) cells. Ongoing studies are addressing the following questions (1) what are the effects of exogenous agents known to modulate CFTR expression on these cell lines; (2) can we identify CFTR expression from the YAC DNA in human cells by mutagenesis of the 3 prime-untranslated region of the CFTR gene; (3) can we use these CFTR YAC vectors to delineate the the cis-acting elements that regulate CFTR expression?
