Newly Designed DNA Fragments for Gene Correction

Abstract

mutations, such as activated oncogenes, could be suitable subjects for gene correction, making it a highly attractive therapeutic strategy. In the small-fragment homologous replacement (SFHR) approach to gene correction, a heat-denatured double-stranded (ds) PCR fragment that is hundreds of bp long and contains the normal sequence is used (Richardson et al. 2002). This method has been utilized with mutations in the CFTR (Kunzelmann et al. 1996,; Goncz et al. 1998, 2001) and dystrophin (Kapsa et al. 2001) genes, and partial gene corrections were obtained. The current SFHR method yields a low correction efficiency, and further improvements are needed. To overcome the limitations of the current SFHR method, we have developed new DNA fragments for gene correction. The first type is a ds DNA fragment prepared by restriction enzyme digestion of plasmid DNA isolated from Escherichia coli. The other type is a single-stranded (ss) DNA fragment prepared by restriction enzyme digestion of ss phagemid DNAs. SFHR-mediated gene correction efficiency was enhanced using these ds and ss DNA fragments. This chapter focuses on the preparation of these DNA fragments for use in SFHRmediated gene correction. The correction of an inactivated Hyg-EGFP gene (fusion gene of hygromycin resistance and enhanced green fluorescence protein genes) serves as a model system (Fig. 1). In this system, codon 34 of the inactive Hyg-EGFP gene (TGA, termination codon) is the target, and its conversion to the TCA sequence (Ser) NGT304 4/23/05 12:40 PM Page 315