title: Stable integration of large (>100 kb) PAC constructs in HaCaT keratinocytes using an integrin-targeting peptide delivery system.
abstract: 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.
authors.affiliation: The Wellcome Trust Centre for Human Genetics, University of Oxford, UK.
authors.forename: S H
authors.initials: SH
authors.lastname: Compton
authors.forename: S
authors.initials: S
authors.lastname: Mecklenbeck
authors.forename: J E
authors.initials: JE
authors.lastname: Mejía
authors.forename: S L
authors.initials: SL
authors.lastname: Hart
authors.forename: M
authors.initials: M
authors.lastname: Rice
authors.forename: R
authors.initials: R
authors.lastname: Cervini
authors.forename: Y
authors.initials: Y
authors.lastname: Barrandon
authors.forename: Z
authors.initials: Z
authors.lastname: Larin
authors.forename: E R
authors.initials: ER
authors.lastname: Levy
authors.forename: L
authors.initials: L
authors.lastname: Bruckner-Tuderman
authors.forename: A
authors.initials: A
authors.lastname: Hovnanian
mesh_terms: D000900:Anti-Bacterial Agents; D015139:Blotting, Southern; D015153:Blotting, Western; D002460:Cell Line; D003094:Collagen / Q000235:genetics*; D004247:DNA / Q000008:administration & dosage*; D004352:Drug Resistance, Microbial / Q000235:genetics; D015870:Gene Expression; D018390:Gene Targeting; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D016023:Integrins / Q000235:genetics; D015603:Keratinocytes / Q000378:metabolism*; D008081:Liposomes; D008164:Luminescent Proteins / Q000235:genetics; D011691:Puromycin; D019030:Receptors, Vitronectin; D012871:Skin Diseases / Q000628:therapy; D012965:Sodium Chloride; D014162:Transfection / Q000379:methods*
chemical_list: D000900:Anti-Bacterial Agents; D016023:Integrins; D008081:Liposomes; D008164:Luminescent Proteins; D019030:Receptors, Vitronectin; C094340:integrin alphavbeta1; D049452:Green Fluorescent Proteins; D012965:Sodium Chloride; D011691:Puromycin; D003094:Collagen; D004247:DNA