title: Small Fragment Homologous Replacement (SFHR): sequence-specific modification of genomic DNA in eukaryotic cells by small DNA fragments.
abstract: The sequence-specific correction of a mutated gene (e.g., point mutation) by the Small Fragment Homologous Replacement (SFHR) method is a highly attractive approach for gene therapy. Small DNA fragments (SDFs) were used in SFHR to modify endogenous genomic DNA in both human and murine cells. The advantage of this gene targeting approach is to maintain the physiologic expression pattern of targeted genes without altering the regulatory sequences (e.g., promoter, enhancer), but the application of this technique requires the knowledge of the sequence to be targeted. In our recent study, an optimized SFHR protocol was used to replace the eGFP mutant sequence in SV-40-transformed mouse embryonic fibroblast (MEF-SV40), with the wild-type eGFP sequence. Nevertheless in the past, SFHR has been used to correct several mutant genes, each related to a specific genetic disease (e.g., spinal muscular atrophy, cystic fibrosis, severe combined immune deficiency). Several parameters can be modified to optimize the gene modification efficiency, as described in our recent study. In this chapter we describe the main guidelines that should be followed in SFHR application, in order to increase technique efficiency.
authors.affiliation: Medical Genetics Section, Department of Biomedicine and Prevention, School of Medicine, Tor Vergata University, Rome, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Luchetti
authors.forename: Arianna
authors.initials: A
authors.lastname: Malgieri
authors.forename: Federica
authors.initials: F
authors.lastname: Sangiuolo
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002453:Cell Cycle; D004247:DNA; D005057:Eukaryotic Cells / Q000378:metabolism*; D005434:Flow Cytometry; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D060005:Genotyping Techniques; D059765:Homologous Recombination; D006801:Humans; D051379:Mice; D017422:Sequence Analysis, DNA; D014162:Transfection
chemical_list: D004247:DNA