Cystic fibrosis is the most common inherited disease in the Caucasian population. About 70% of all CF chromosomes carry the DeltaF508 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 DeltaF508 locus of endogenous CFTR was achieved by small fragment homologous replacement (SFHR). Transformed human airway epithelial cells (CFBE41o(-)), homozygous for DeltaF508 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 approximately 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 DeltaF alleles. DNA fingerprint analysis confirmed that the colonies were isogenic and derived from the parental CFBE41o(-) 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(-)) and mutant (CFBE41o(-)) cell lines, indicated that the allele-specific PCR was at least 25-fold more sensitive than non allele-specific PCR. These results suggest that the colony is not yet clonal, but still contains a population of parental, CFBE41o(-) 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 population. Nonallele-specific reverse transcriptase PCR (RT-PCR) analysis of the CFTR mRNA indicated that two of the colonies expressed both WT and DeltaF508 CFTR mRNA, while one colony appeared to express only the WT mRNA. The mRNA results were confirmed by sequence analysis of 3' end primer extension products from the mRNA of CFTR exon 10 showing that the mRNA containing exon 10. Furthermore, a survey of primer extension products indicated no random insertion of the fragment in an expressed gene. This study demonstrates SFHR-mediated modification of the DeltaF508 allele in DeltaF508 homozygote human airway epithelial cells over multiple generations. The resultant cells express WT-CFTR mRNA and can be subcloned further to isolate isogenic clonal populations of cells.
Bruscia, E., Sangiuolo, F., Sinibaldi, P., Goncz, K.k., Novelli, G., & Gruenert, D.c. (2002). Isolation of CF cell lines corrected at ΔF508-CFTR locus by SFHR-mediated targeting. GENE THERAPY, 9(11), 683-685.
|Tipologia:||Articolo su rivista|
|Citazione:||Bruscia, E., Sangiuolo, F., Sinibaldi, P., Goncz, K.k., Novelli, G., & Gruenert, D.c. (2002). Isolation of CF cell lines corrected at ΔF508-CFTR locus by SFHR-mediated targeting. GENE THERAPY, 9(11), 683-685.|
|IF:||Con Impact Factor ISI|
|Settore Scientifico Disciplinare:||Settore MED/03 - Genetica Medica|
|Revisione (peer review):||Esperti anonimi|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1038/sj.gt.3301741|
|Stato di pubblicazione:||Pubblicato|
|Data di pubblicazione:||2002|
|Titolo:||Isolation of CF cell lines corrected at ΔF508-CFTR locus by SFHR-mediated targeting|
|Autori:||Bruscia, E; Sangiuolo, F; Sinibaldi, P; Goncz, Kk; Novelli, G; Gruenert, Dc|
|Appare nelle tipologie:||01 - Articolo su rivista|