Please use this identifier to cite or link to this item:
Title: Escherichia coli-cloned CFTR loci relevant for human artificial chromosome therapy
Authors: Rocchi, Lucia
Braz, Carla
Cattani, Sonja
Ramalho, Anabela
Christan, Sulith
Edlinger, Marlene
Ascenzioni, Fiorentina
Laner, Andreas
Kraner, Simone
Amaral, Margarida
Schindelhauer, Dirk
Keywords: Doenças Genéticas
Issue Date: Sep-2010
Publisher: Mary Ann Liebert
Citation: Hum Gene Ther. 2010 Sep;21(9):1077-92
Abstract: Classical gene therapy for cystic fibrosis has had limited success because of immune response against viral vectors and short-term expression of cDNA-based transgenes. These limitations could be overcome by delivering the complete genomic CFTR gene on nonintegrating human artificial chromosomes (HACs). Here, we report reconstruction of the genomic CFTR locus and analyze incorporation into HACs of three P1 phage-based and F factor bacteria-based artificial chromosomes (PACs/BACs) of various sizes: (1) 5A, a large, nonselectable BAC containing the entire wild-type CFTR locus extending into both adjacent genes (296.8-kb insert, from kb -58.4 to +51.4) containing all regulators; (2) CGT21, a small, selectable, telomerized PAC (134.7 kb, from kb -60.7 to + 2) containing a synthetic last exon joining exon 10, EGFP, exon 24, and the 3' untranslated region; and (3) CF225, a midsized, nonselectable PAC (225.3 kb, from kb -60.7 to +9.8) ligated from two PACs with optimized codons and a silent XmaI restriction variant to discriminate transgene from endogenous expression. Cotransfection with telomerized, blasticidin-S-selectable, centromere-proficient α-satellite constructs into HT1080 cells revealed a workable HAC formation rate of 1 per ∼25 lines when using CGT21 or 5A. CF225 was not incorporated into a de novo HAC in 122 lines analyzed, but integrants were expressed. Stability analyses suggest the feasibility of prefabricating a large, tagged CFTR transgene that stably replicates in the proximity of a functional centromere. Although definite conclusions about HAC-proficient construct configurations cannot be drawn at this stage, important transfer resources were generated and characterized, demonstrating the promise of de novo HACs as potentially ideal gene therapy vector systems.
Peer review: yes
ISSN: 1043-0342
Publisher Version:
Appears in Collections:DGH - Artigos em revistas internacionais

Files in This Item:
File Description SizeFormat 
Escherichia coli-cloned CFTR loci relevant for human artificial.pdf680,49 kBAdobe PDFView/Open    Request a copy

FacebookTwitterDeliciousLinkedInDiggGoogle BookmarksMySpace
Formato BibTex MendeleyEndnote Degois 

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.