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Titolo:
Targeted correction of a defective selectable marker gene in human epithelial cells by small DNA fragments
Autore:
Colosimo, A; Goncz, KK; Novelli, G; Dallapiccola, B; Gruenert, DC;
Indirizzi:
Univ Vermont, Colchester Res Facil, Dept Med, Human Mol Genet Unit, Colchester, VT 05446 USA Univ Vermont Colchester VT USA 05446 Genet Unit, Colchester, VT 05446 USA Univ Chieti G DAnnunzio, Dept Biomed Sci, Chieti, Italy Univ Chieti G DAnnunzio Chieti Italy io, Dept Biomed Sci, Chieti, Italy Univ Rome Tor Vergata, Dept Biopathol & Diagnost Imaging, Genet Sect, Rome, Italy Univ Rome Tor Vergata Rome Italy gnost Imaging, Genet Sect, Rome, Italy Univ Rome La Sapienza, Dept Expt Med & Pathol, Rome, Italy Univ Rome La Sapienza Rome Italy a, Dept Expt Med & Pathol, Rome, Italy CSS Mendel Inst, Rome, Italy CSS Mendel Inst Rome ItalyCSS Mendel Inst, Rome, Italy
Titolo Testata:
MOLECULAR THERAPY
fascicolo: 2, volume: 3, anno: 2001,
pagine: 178 - 185
SICI:
1525-0016(200102)3:2<178:TCOADS>2.0.ZU;2-S
Fonte:
ISI
Lingua:
ENG
Soggetto:
MAMMALIAN-CELLS; RECOMBINATION; REPAIR; OLIGONUCLEOTIDE; SEQUENCES; BARRIER; THERAPY; BREAKS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
29
Recensione:
Indirizzi per estratti:
Indirizzo: Gruenert, DC Univ Vermont, Colchester Res Facil, Dept Med, Human Mol GenetUnit, 203 S Park Dr,Suite 2, Colchester, VT 05446 USA Univ Vermont 203 S Park Dr,Suite 2 Colchester VT USA 05446 SA
Citazione:
A. Colosimo et al., "Targeted correction of a defective selectable marker gene in human epithelial cells by small DNA fragments", MOL THER, 3(2), 2001, pp. 178-185

Abstract

A novel gene targeting strategy, small fragment homologous replacement (SFHR), has been used to correct specific genomic lesions in human epithelial cells. The frequency of targeting was estimated to be 1-10%. However, giventhe genomic target, the cystic fibrosis transmembrane conductance regulator (CFTR) gene, it is difficult to accurately quantify targeting frequency. As an alternative to targeting CFTR, targeted correction of a mutant selectable marker or reporter gene would be more amenable to accurate and rapid quantification of gene targeting efficiency. The present study evaluates theconditions that modulate SFHR-mediated correction of a defective Zeocin antibiotic resistance (Zeo(r)) gene that has been inactivated by a 4-bp insertion. The conditions include delivery systems, plasmid-to-fragment ratio, fragment length, and fragment strandedness (single- or double-stranded DNA). Targeting fragments comprise the wild-type Zeo(r) gene sequence and were either 410 (Zeo1) or 458 bp (Zeo3). Expression vectors containing the corrected Zeo(r) gene were isolated as episomal plasmids or were allowed to stably integrate into cultured human airway epithelial cells. Correction of the Zeo(r) gene was phenotypically defined as restoration of resistance to Zeocin in either bacteria or epithelial cell clones. Extrachromosomal gene correction was assayed using polymerase chain reaction amplification, restriction enzyme digestion, DNA sequencing, and Southern blot hybridization analysis of DNA from isolated prokaryotic and eukaryotic clones. Neither random sequence alteration in the target episomal gene nor random integration of the small fragments was detected. Targeted correction efficiencies of up to 4% were attained. These studies provide insight into parameters that can be modulated for the optimization of SFHR-mediated targeting.

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Documento generato il 02/04/20 alle ore 09:27:15