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Isolation of CF cell lines corrected at δF508-CFTR locus by SFHR-mediated targeting.
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- Author(s): Bruscia, E.; Sangiuolo, F.; Sinibaldi, P.; Goncz, K.K.; Novelli, G.; Gruenert, D.C.
- Source:
Gene Therapy; 6/1/2002, Vol. 9 Issue 11, p683, 3p- Subject Terms:
- Source:
- Additional Information
- Abstract: Cystic fibrosis is the most common inherited disease in the Caucasian population. About 70% of all CF chromosomes carry the ΔF508 mutation, a 3-bp deletion that results in the loss of a phenylalanine at amino acid 508 in the CF transmembrane conductance regulator (CFTR) protein. Direct modification of the ΔF508 locus of endogenous CFTR was achieved by small fragment homologous replacement (SFHR). Transformed human airway epithelial cells (CFBE41o[sup -]), homozygous for ΔF508 mutation, were transfected with small fragments (491-bp) of wild-type (WT) CFTR DNA comprising exon 10 and the flanking introns. The DNA fragments were in a liposome-DNA complex at a charge ratio of 6:1 (+:-), respectively). The population of transfected cells was subcloned by limiting dilution at ∼1 cell/well in 96-well plates. Individual colonies were isolated and analyzed. The DNA from several colonies was characterized by radiolabeled, nonallele-specific and radiolabeled, allele-specific PCR amplification, as well as by genomic DNA fingerprinting. The CFTR-WT allele was detected in five of these colonies by allele-specific PCR amplification thus indicating that the cell lines carried both WT and ΔF alleles. DNA fingerprint analysis confirmed that the colonies were isogenic and derived from the parental CFBE41o[sup -] cell line. Although, the WT allele was detected by allele-specific PCR, it was not detected initially when the same samples were analyzed by non allele-specific PCR. A sensitivity assay, mixing the genomic DNA of wild-type (16HBE14o[sup -]) and mutant (CFBE41o[sup -]) cell lines, indicated that the allelespecific PCR was at least 25-fold more sensitive than nonallele-specific PCR. These results suggest that the colony is not yet clonal, but still contains a population of parental, CFBE41o[sup -] cells that have not been modified. Based on the mixing analysis, the proportion of corrected cells appears to be between 1 and 10% of the total... [ABSTRACT FROM AUTHOR]
- Abstract: Copyright of Gene Therapy is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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