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The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR) Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes

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 wi...

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Autores principales: Pierandrei, Silvia, Luchetti, Andrea, Sanchez, Massimo, Novelli, Giuseppe, Sangiuolo, Federica, Lucarelli, Marco
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014528/
https://www.ncbi.nlm.nih.gov/pubmed/27045208
http://dx.doi.org/10.1038/mtna.2016.2
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author Pierandrei, Silvia
Luchetti, Andrea
Sanchez, Massimo
Novelli, Giuseppe
Sangiuolo, Federica
Lucarelli, Marco
author_facet Pierandrei, Silvia
Luchetti, Andrea
Sanchez, Massimo
Novelli, Giuseppe
Sangiuolo, Federica
Lucarelli, Marco
author_sort Pierandrei, Silvia
collection PubMed
description 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.
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spelling pubmed-50145282016-09-19 The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR) Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes Pierandrei, Silvia Luchetti, Andrea Sanchez, Massimo Novelli, Giuseppe Sangiuolo, Federica Lucarelli, Marco Mol Ther Nucleic Acids Original Article 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. Nature Publishing Group 2016-04 2016-04-05 /pmc/articles/PMC5014528/ /pubmed/27045208 http://dx.doi.org/10.1038/mtna.2016.2 Text en Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy http://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/
spellingShingle Original Article
Pierandrei, Silvia
Luchetti, Andrea
Sanchez, Massimo
Novelli, Giuseppe
Sangiuolo, Federica
Lucarelli, Marco
The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR) Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes
title The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR) Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes
title_full The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR) Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes
title_fullStr The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR) Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes
title_full_unstemmed The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR) Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes
title_short The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR) Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes
title_sort gene targeting approach of small fragment homologous replacement (sfhr) alters the expression patterns of dna repair and cell cycle control genes
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014528/
https://www.ncbi.nlm.nih.gov/pubmed/27045208
http://dx.doi.org/10.1038/mtna.2016.2
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