Copy number-dependent expression of a YAC-cloned human CFTR gene in a human epithelial cell line.
Initial studies for gene therapy of cystic fibrosis have used heterologous promoters to drive expression of the cystic fibrosis transmembrane regulator (CFTR) cDNA. An alternative approach would be to have constructs based on the endogenous promoter which could give tissue-specific and normal, regulated levels of expression. The DNA elements necessary for this remain unidentified so we have investigated CFTR expression from a 310 kb yeast artificial chromosome (YAC) clone which contains the intact human CFTR gene consisting of 27 exons spread over 230 kb of genomic DNA and also about 50 kb of 5' DNA. In order to distinguish the gene on the YAC, a restriction site change was introduced into the 3' untranslated region of the gene in the YAC. The YAC was then transferred into the human colonic adenocarcinoma cell line Caco-2 which expresses endogenous CFTR. The CFTR gene on the YAC was found to be well expressed in the Caco-2 cells and, in the two cell lines analysed, the ratio of YAC-derived mRNA to endogenous mRNA was the same as the ratio of YAC DNA to endogenous DNA. This indicates that each copy of the CFTR gene on a YAC integrated in the Caco-2 cells is being expressed at approximately the same level as each of the endogenous genes. Thus, the YAC clone contains all the long-range elements necessary to confer full levels of expression, independent of position of integration, in human Caco-2 cells. Deletion of this YAC clone should allow the elucidation of the DNA elements controlling human CFTR expression and the development of constructs for gene therapy.
Vassaux
G
G
Imperial College School of Medicine at St Mary's, London, UK.
Manson
A L
AL
Huxley
C
C
D018938:Caco-2 Cells; D018244:Chromosomes, Artificial, Yeast; D003550:Cystic Fibrosis / Q000628:therapy*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D017353:Gene Deletion; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction; D014162:Transfection
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator