Stable integration of large (>100 kb) PAC constructs in HaCaT keratinocytes using an integrin-targeting peptide delivery system

Détails

ID Serval
serval:BIB_099209ADE475
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
Stable integration of large (>100 kb) PAC constructs in HaCaT keratinocytes using an integrin-targeting peptide delivery system
Périodique
Gene Therapy
Auteur(s)
Compton  S. H., Mecklenbeck  S., Mejia  J. E., Hart  S. L., Rice  M., Cervini  R., Barrandon  Y., Larin  Z., Levy  E. R., Bruckner-Tuderman  L., Hovnanian  A.
ISSN
0969-7128 (Print)
Statut éditorial
Publié
Date de publication
09/2000
Volume
7
Numéro
18
Pages
1600-5
Notes
Journal Article
Research Support, Non-U.S. Gov't --- Old month value: Sep
Résumé
Transfer of large DNA constructs in gene therapy studies is being recognised for its importance in maintaining the natural genomic environment of the gene of interest and providing tissue-specific regulation and control. However, methods used to deliver such constructs have been poorly studied. We used a receptor-mediated, integrin-targeting transfection system enhanced by liposomes, to deliver a 110 kb PAC (P1-based artificial chromosome) to HaCaT keratinocytes. The PAC contained the collagen VII locus, an EGFP (enhanced green fluorescent protein) reporter gene and the puromycin resistance gene (pac) to allow selection of stably transfected cells. Analysis of puromycin resistant and EGFP-expressing colonies by Western blot showed that collagen VII production increased dramatically after transfection, indicating successful transfer of a large fully functional genomic locus. Fluorescent in situ hybridisation (FISH) and Southern blot analysis revealed that the PAC had integrated as at least one copy per cell. EGFP expression has persisted for 35 weeks, suggesting stable transgene expression. We conclude that the integrin-targeting peptide method of gene delivery is an effective means of stably delivering large DNA constructs to human keratinocytes and could be of benefit for genomic gene therapy approaches.
Mots-clé
Anti-Bacterial Agents Blotting, Southern Blotting, Western Cell Line Collagen/*genetics DNA/*administration & dosage Drug Resistance, Microbial/genetics Gene Expression Gene Targeting Gene Therapy/*methods Genetic Vectors Green Fluorescent Proteins Humans In Situ Hybridization, Fluorescence Integrins/genetics Keratinocytes/*metabolism Liposomes Luminescent Proteins/genetics Puromycin *Receptors, Vitronectin Skin Diseases/therapy Sodium Chloride Transfection/*methods
Pubmed
Web of science
Open Access
Oui
Création de la notice
28/01/2008 8:41
Dernière modification de la notice
20/08/2019 12:31
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