Hiroyuki Tsuchiya1, Hideyoshi Harashima, Hiroyuki Kamiya. 1. Laboratory for Molecular Design of Pharmaceutics and COE Program in the 21st Century, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Sapporo, Hokkaido 060-0812, Japan.
Abstract
BACKGROUND: The correction of a mutated gene by the small fragment homologous replacement (SFHR) method is a highly attractive approach for gene therapy. However, the current SFHR method with a heat-denatured double-stranded PCR fragment yielded a low correction efficiency. METHODS: Single-stranded (ss) DNA fragments were prepared from ss phagemid DNA and tested in a gene correction assay with an inactivated Hyg-EGFP fusion gene, as a model target. RESULTS: A 606-nt sense, ss DNA fragment dramatically (12-fold) improved the gene correction efficiency, although the antisense strand showed only minimal correction efficiency. CONCLUSIONS: These results suggest that the use of a sense, single-stranded DNA fragment is useful in the SFHR method for the correction of mutated genes. Copyright (c) 2004 John Wiley & Sons, Ltd.
BACKGROUND: The correction of a mutated gene by the small fragment homologous replacement (SFHR) method is a highly attractive approach for gene therapy. However, the current SFHR method with a heat-denatured double-stranded PCR fragment yielded a low correction efficiency. METHODS: Single-stranded (ss) DNA fragments were prepared from ss phagemid DNA and tested in a gene correction assay with an inactivated Hyg-EGFP fusion gene, as a model target. RESULTS: A 606-nt sense, ss DNA fragment dramatically (12-fold) improved the gene correction efficiency, although the antisense strand showed only minimal correction efficiency. CONCLUSIONS: These results suggest that the use of a sense, single-stranded DNA fragment is useful in the SFHR method for the correction of mutated genes. Copyright (c) 2004 John Wiley & Sons, Ltd.
Authors: Nanna M Jensen; Trine Dalsgaard; Maria Jakobsen; Roni R Nielsen; Charlotte B Sørensen; Lars Bolund; Thomas G Jensen Journal: J Biomed Sci Date: 2011-02-02 Impact factor: 8.410