zed below in abstract form: 1. Construction of a yac vector to study CFTR gene expression. The cis-acting elements that regulate CFTR gene expression are poorly characterized. To date regulatory studies have relied on small reporter gene constructs to define the minimal promoter region producing conflicting results. One explaination for these discrepancies is that additional regulatory regions are present at distant sites which have not been included in the vectors studied to date. In addition, several vectors have not included 24 bp immediately upstream from the start site of CFTR transcription. Therefore, construction of a vector that can incorporate a more complete representation of CFTR flanking DNA may aid in identifying cis-acting elements involved in the regulation of CFTR gene expression. YAC vectors are ideally suited for these regulatory studies since they can incorporate large fragments of DNA that can easily be manipulated in yeast before being transferred into mammalian cells. Previous studies in our laboratory have demonstrated that a 610kb YAC (y1/8/7-R) contains a functional CFTR gene (Hum. Mol. Genet. 1997,6:59-68). We have used ~335kb of flanking DNA upstream from the CFTR gene from y1/8/7-R to drive the expression of a luciferase reporter gene. A new YAC was created by replacing the right arm of y1/8/7- R with a new arm containing genes for luciferase and neomycin resistance (neor) driven by the mouse phosphoglycerate kinase-1 promoter and the yeast selectable marker LYS2. This replacement was accomplished by incorporating a region of homology at the start site of CFTR transcription into the vector. The resulting YAC contains the luciferase gene driven by ~335kb CFTR 5?- flanking DNA. In addition, a CFTR intron 1 fragment, reported to enhance CFTR gene expression in vitro, was incorporated into this YAC. A second YAC will be created without this intron element. Yeast containing the modified YACs were identified by their ability to grown on media lacking lysine. The DNA from these yeast was analyzed by pulsed field gel electrophoresis and Southern hybridization using neor and luciferase probes. Analysis of appropriately sized YACs by long range restriction mapping confirmed that no gross rearrangement of the CFTR flanking DNA had taken place. In addition, the site of recombination and the introduced intron 1 fragment were sequenced and confirmed to be correct. These YACs were transferred into mammalian cells by spheroplast fusion to derive stable transformants. Purified YAC DNA will also be transfected into mammalian cells allowing for both transient and long-term analyses. We will initially use chromosomal fragmentation to study the of the role of flanking DNA in the regulation of CFTR expression. Later studies will focus on elements of the CFTR 5?- flanking DNA identified to be important by fragmentation. More detailed analysis will be performed by subcloning these regions into appropriate vectors for further study. 2. Expression of a full-length, uniquely-identifiable CFTR gene from a 610 kb YAC stably introduced into a CF airway epithelial cell line. The tight spatial, temporal and quantitative control of CFTR expression in vivo, as well as data suggesting negative consequences of CFTR overexpression in vitro, have implications for CFTR gene therapy strategies. One approach to generate physiologic levels of CFTR expression is to construct vectors which include the CFTR promoter, and regulatory elements which control the endogenous expression of this 230 kb gene. However, the DNA capacity of most commonly used vectors is limited to little more than the 4.7 kb CFTR cDNA driven by a heterologous promoter. Our goal was to develop a physiologically-relevant CFTR vector using the large DNA cloning capacity of a yeast artificial chromosome (YAC) and to demonstrate its ability to achieve phenotype correction in CF airway epithelial cells. The YAC that we started with contains a full-length, functional copy of CFTR (Mogayzel et al., Hum. Mol. Gen. 6:59-68, 1997). This YAC was chosen because it contains ~ 375kb of DNA flanking CFTR, potentially important, given that the specific cis-acting CFTR regulatory elements are not yet well-defined. This YAC was retrofitted with a neomycin resistance gene (NeoR), and then modified to enable YAC-derived CFTR mRNA to be distinguished in human cells from endogenous CFTR mRNA. This YAC modification was accomplished in yeast using site-directed mutagenesis and two- step gene-replacement to generate two new restriction sites in the 3? untranslated region (UTR) of exon 24. Preliminary data in CHO cells demonstrated that this modified YAC expressed full- length mRNA and functional CFTR. Gel-purified YAC DNA was stably introduced into CFT1 cells, an immortalized human airway epithelial cell line with a DF508/DF508 genotype (Yankaskas et al., AJP-Cell 264:C1219-1230, 1993), by lipid-mediated transfection. As expected using human cells, stable YAC transfection efficiency was low (1 NeoR clone per ~5 x105 cells) and critically dependent upon the integrity and purity of the DNA. The NeoR CFT1 clones were screened by PCR for DNA located in the right YAC arm (~600 kb downstream from Neo). Clones positive for the right YAC arm were evaluated for CFTR expression using RT-PCR and restriction enzyme analysis based on the 3?-UTR mutations. The results demonstrate the presence of YAC-derived CFTR mRNA and the absence of endogenous CFTR mRNA in the CFT1 cell line. To our knowledge, this is the first successful stable transfer of CFTR under the control of its own regulatory elements into a CF epithelial cell line. We are currently determining the integration site and copy number of this YAC and most importantly, the ability of CFTR expressed from the YAC to correct the CF phenotype in these cells. This study sets the stage for future analysis of the feasibility and physiologic relevance of gene transfer in human cells using CFTR under the control of regulatory elements which mimics endogenous CFTR expression.
