CFTR intron I increases luciferase expression driven by CFTR 5'-flanking DNA in a yeast artificial chromosome

Peter J. Mogayzel, Melissa A. Ashlock

Research output: Contribution to journalArticle

Abstract

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 ~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 I 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. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)211-215
Number of pages5
JournalGenomics
Volume64
Issue number2
DOIs
StatePublished - Mar 1 2000

ASJC Scopus subject areas

  • Genetics

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