title: The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR) Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes.
abstract: Cellular responses and molecular mechanisms activated by exogenous DNA that invades cells are only partially understood. This limits the practical use of gene targeting strategies. Small fragment homologous replacement (SFHR) uses a small exogenous wild-type DNA fragment to restore the endogenous wild-type sequence; unfortunately, this mechanism has a low frequency of correction. In this study, we used a mouse embryonic fibroblast cell line with a stably integrated mutated gene for enhanced green fluorescence protein. The restoration of a wild-type sequence can be detected by flow cytometry analysis. We quantitatively analyzed the expression of 84 DNA repair genes and 84 cell cycle control genes. Peculiar temporal gene expression patterns were observed for both pathways. Different DNA repair pathways, not only homologous recombination, as well as the three main cell cycle checkpoints appeared to mediate the cellular response. Eighteen genes were selected as highly significant target/effectors of SFHR. We identified a wide interconnection between SFHR, DNA repair, and cell cycle control. Our results increase the knowledge of the molecular mechanisms involved in cell invasion by exogenous DNA and SFHR. Specific molecular targets of both the cell cycle and DNA repair machineries were selected for manipulation to enhance the practical application of SFHR.
authors.affiliation: Department of Cellular Biotechnologies and Hematology, Sapienza University of Rome, Rome, Italy.|Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Pierandrei
authors.affiliation: Department of Biomedicine and Prevention, Tor Vergata University, Rome, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Luchetti
authors.affiliation: Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Sanchez
authors.affiliation: Department of Biomedicine and Prevention, Tor Vergata University, Rome, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Novelli
authors.affiliation: Department of Biomedicine and Prevention, Tor Vergata University, Rome, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Sangiuolo
authors.affiliation: Department of Cellular Biotechnologies and Hematology, Sapienza University of Rome, Rome, Italy.|Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Lucarelli