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In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique
BACKGROUND: SFHR (small fragment homologous replacement)-mediated targeting is a process that has been used to correct specific mutations in mammalian cells. This process involves both chemical and cellular factors that are not yet defined. To evaluate potential of this technique for gene therapy it...
Autores principales: | , , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2002
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC130050/ https://www.ncbi.nlm.nih.gov/pubmed/12243649 http://dx.doi.org/10.1186/1471-2350-3-8 |
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author | Sangiuolo, Federica Bruscia, Emanuela Serafino, Annalucia Nardone, Anna Maria Bonifazi, Emanuela Lais, Monica Gruenert, Dieter C Novelli, Giuseppe |
author_facet | Sangiuolo, Federica Bruscia, Emanuela Serafino, Annalucia Nardone, Anna Maria Bonifazi, Emanuela Lais, Monica Gruenert, Dieter C Novelli, Giuseppe |
author_sort | Sangiuolo, Federica |
collection | PubMed |
description | BACKGROUND: SFHR (small fragment homologous replacement)-mediated targeting is a process that has been used to correct specific mutations in mammalian cells. This process involves both chemical and cellular factors that are not yet defined. To evaluate potential of this technique for gene therapy it is necessary to characterize gene transfer efficacy in terms of the transfection vehicle, the genetic target, and the cellular processing of the DNA and DNA-vehicle complex. METHODS: In this study, small fragments of genomic cystic fibrosis (CF) transmembrane conductance regulator (CFTR) DNA, that comprise the wild-type and ΔF508 sequences, were transfected into immortalized CF and normal airway epithelial cells, respectively. Homologous replacement was evaluated using PCR and sequence-based analyses of cellular DNA and RNA. Individual stages of cationic lipid-facilitated SFHR in cultured cell lines were also examined using transmission electron microscopy (TEM). RESULTS: We demonstrated that the lipid/DNA (+/-) ratio influences the mode of entry into the cell and therefore affects the efficacy of SFHR-mediated gene targeting. Lipid/DNA complexes with more negative ratios entered the cell via a plasma membrane fusion pathway. Transfer of the DNA that relies on an endocytic pathway appeared more effective at mediating SFHR. In addition, it was also clear that there is a correlation between the specific cell line transfected and the optimal lipid/DNA ratio. CONCLUSIONS: These studies provide new insights into factors that underlie SFHR-mediated gene targeting efficacy and into the parameters that can be modulated for its optimization. |
format | Text |
id | pubmed-130050 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2002 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-1300502002-10-25 In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique Sangiuolo, Federica Bruscia, Emanuela Serafino, Annalucia Nardone, Anna Maria Bonifazi, Emanuela Lais, Monica Gruenert, Dieter C Novelli, Giuseppe BMC Med Genet Research Article BACKGROUND: SFHR (small fragment homologous replacement)-mediated targeting is a process that has been used to correct specific mutations in mammalian cells. This process involves both chemical and cellular factors that are not yet defined. To evaluate potential of this technique for gene therapy it is necessary to characterize gene transfer efficacy in terms of the transfection vehicle, the genetic target, and the cellular processing of the DNA and DNA-vehicle complex. METHODS: In this study, small fragments of genomic cystic fibrosis (CF) transmembrane conductance regulator (CFTR) DNA, that comprise the wild-type and ΔF508 sequences, were transfected into immortalized CF and normal airway epithelial cells, respectively. Homologous replacement was evaluated using PCR and sequence-based analyses of cellular DNA and RNA. Individual stages of cationic lipid-facilitated SFHR in cultured cell lines were also examined using transmission electron microscopy (TEM). RESULTS: We demonstrated that the lipid/DNA (+/-) ratio influences the mode of entry into the cell and therefore affects the efficacy of SFHR-mediated gene targeting. Lipid/DNA complexes with more negative ratios entered the cell via a plasma membrane fusion pathway. Transfer of the DNA that relies on an endocytic pathway appeared more effective at mediating SFHR. In addition, it was also clear that there is a correlation between the specific cell line transfected and the optimal lipid/DNA ratio. CONCLUSIONS: These studies provide new insights into factors that underlie SFHR-mediated gene targeting efficacy and into the parameters that can be modulated for its optimization. BioMed Central 2002-09-23 /pmc/articles/PMC130050/ /pubmed/12243649 http://dx.doi.org/10.1186/1471-2350-3-8 Text en Copyright © 2002 Sangiuolo et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. |
spellingShingle | Research Article Sangiuolo, Federica Bruscia, Emanuela Serafino, Annalucia Nardone, Anna Maria Bonifazi, Emanuela Lais, Monica Gruenert, Dieter C Novelli, Giuseppe In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique |
title | In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique |
title_full | In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique |
title_fullStr | In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique |
title_full_unstemmed | In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique |
title_short | In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique |
title_sort | in vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (sfhr) technique |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC130050/ https://www.ncbi.nlm.nih.gov/pubmed/12243649 http://dx.doi.org/10.1186/1471-2350-3-8 |
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