CFTR intron 1 increases luciferase expression driven by CFTR 5'-flanking DNA in a yeast artificial chromosome.
The DNA elements that account for the highly regulated expression of the cystic fibrosis transmembrane conductance regulator gene (CFTR) are poorly understood. The goal of this study was to assess the feasibility of using a yeast artificial chromosome (YAC)-based reporter gene construct to define these elements further. An approximately 350-kb YAC (y5'luc) was constructed by replacing CFTR with a luciferase reporter gene (luc). A second YAC (y5'lucI) was similarly constructed but included a putative positive regulatory element from CFTR intron 1. Stable Chinese hamster ovary (CHO-K1) cell clones were derived using each YAC to assess the role that luc copy number and the presence of intron 1 played in luc expression. The CHO-K1 clonal cell lines demonstrated a wide range of luciferase activity. On average, this activity was significantly higher in clones derived from y5'lucI. After correcting for luc copy number, the presence of intron 1 was still associated with an increase in luciferase activity (P < 0.05), despite the fact that luciferase activity did not correlate with luc copy number in y5'luc-derived clones (r = -0.12). In contrast, the luciferase activity correlated well with luc copy number in the clones derived from y5'luc (r = 0. 75). These data are consistent with a positive role for intron 1 in regulating CFTR expression, but suggest that copy number is not the only factor that determines expression levels, particularly when this element is present. This YAC-based reporter system will provide a unique strategy for further assessment of the cis-acting elements that control CFTR expression.
Mogayzel
P J
PJ
Eudowood Division of Pediatric Respiratory Sciences, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287-2533, USA. mogayzel@mail.jhmi.edu
Ashlock
M A
MA
D000818:Animals; D015139:Blotting, Southern; D016466:CHO Cells; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D002999:Clone Cells; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D018628:Gene Dosage; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D007438:Introns / Q000235:genetics / Q000502:physiology*; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology*; D014162:Transfection; D015854:Up-Regulation
D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D008156:Luciferases