Human Molecular Genetics, Vol 7, 1913-1919, Copyright © 1998 by Oxford University Press
KK Goncz, K Kunzelmann, Z Xu and DC Gruenert
Recent studies have reported that mutant genomic cystic fibrosis (CF)
transmembrane conductance regulator ( CFTR ) sequences can be corrected in
transformed CF airway epithelial cell lines by targeted replacement with
small fragments of DNA with wild-type sequence. To determine if the
observed genotype modification following small fragment homologous
replacement (SFHR) was limited to transformed CF cell lines, further
studies were carried out in both transformed and non-transformed primary
normal airway epithelial cells. The endogenous genotype of these normal
cell lines was modified following liposome or dendrimer transfection using
DNA fragments with DeltaF508 CFTR sequence (488 nt, complementary single
strands) designed to also contain a unique restriction enzyme cleavage site
(Xho I). Replacement at the appropriate genomic locus by exogenous
DeltaF508 CFTR DNA and its expression as mRNA was demonstrated by PCR
amplification of genomic DNA and mRNA-derived cDNA as well as Xho I
digestion of the PCR products. These studies show that SFHR occurs in both
transformed and non- transformed primary human airway epithelial cells and
indicate that single base substitution (the silent mutation giving rise to
the Xho I site) and deletion or insertion of at least three consecutive
bases can be achieved in both normal and CF epithelial cells. Furthermore,
these studies reiterate the potential of SFHR as a strategy for a number of
gene targeting applications, such as site-specific mutagenesis, development
of transgenic animals, development of isogenic cell lines and for gene
therapy.
ARTICLES
Targeted replacement of normal and mutant CFTR sequences in human airway epithelial cells using DNA fragments
Cardiovascular Research Institute, Gene Therapy Core Center and Cystic Fibrosis Research Center and Department of Laboratory Medicine and Stomatology, University of California, San Francisco, CA 94143, USA.
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