small fragment homologous replacement evaluation of factors influencing modification efficiency in an eukaryotic assay system小片段同源替代评估影响因素修改一个真核生物测定系统的效率.pdfVIP

Small Fragment Homologous Replacement: Evaluation of Factors Influencing Modification Efficiency in an Eukaryotic Assay System 1. 1. 2 3 3 Andrea Luchetti , Antonio Filareto , Massimo Sanchez , Giampiero Ferraguti , Marco Lucarelli , 4 1 1 Giuseppe Novelli , Federica Sangiuolo *, Arianna Malgieri 1 Department of Biopathology and Diagnostic Imaging, Medical Genetics Section, School of Medicine, Tor Vergata University, Rome, Italy, 2 Department of Cell Biology ` and Neurosciences, Istituto Superiore di Sanita, Rome, Italy, 3 Department of Cellular Biotechnologies and Hematology, Sapienza University of Rome, Rome, Italy, 4 National Agency of Evaluation for University and Research (ANVUR) and Ospedale San Pietro Fatebenefratelli, Rome, Italy Abstract Homologous Replacement is used to modify specific gene sequences of chromosomal DNA in a process referred to as ‘‘Small Fragment Homologous Replacement’’, where DNA fragments replace genomic target resulting in specific sequence changes. To optimize the efficiency of this process, we developed a reporter based assay system where the replacement frequency is quantified by cytofluorimetric analysis following restoration of a stably integrated mutated eGFP gene in the genome of SV-40 immortalized mouse embryonic fibroblasts (MEF-SV-40). To obtain the highest correction frequency with this system, several parameters were considered: fragment synthesis and concentration, cell cycle phase and methylation status of both fragment and recipient genome. In addition, different drugs were employed to test their ability to improve techniq