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 technique efficiency. SFHR-mediated genomic modification resulted to be stably transmitted for several cell generations and confirmed at transcript and genomic levels. Modification efficiency was estimated in a range of 0.01–0.5%, further increasing when PARP-1 repair pathway was inhibited. In this study, for the first time SFHR efficiency issue was systematically approached and in part addressed, therefore opening new potential therapeutic ex-vivo applications.
Highlights
In situ modification by gene targeting approach allows the recovery of a normal gene function [1], offering significant advantages compared to gene augmentation
This hybrid structure could activate the endogenous machinery involved in DNA repair and, by homologous recombination (HR), allow the Small DNA Fragments (SDFs) to be integrated into the genomic DNA [5]
fluorescent in situ hybridization (FISH) analysis on D1 clone demonstrated the genomic integration of the pCEP4/mut-eGFP vector (Fig. S1)
Summary
In situ modification by gene targeting approach allows the recovery of a normal gene function [1], offering significant advantages compared to gene augmentation. Mutated genetic instructions are site- modified in long-term and genetically inheritable manner, maintaining their native sequence context. By this way, targeted gene results modulated by the endogenous regulatory machinery, maintaining physiologic expression pattern. It is likely that the fragment recognizes and anneals to its homologous target, promoting the formation of a D-loop structure. This hybrid structure could activate the endogenous machinery involved in DNA repair and, by HR, allow the SDF to be integrated into the genomic DNA [5]. The SFHR-mediated DNA modification has been shown to properly target genomic DNA in both differentiated and undifferentiated stem cells [18], resulting in long-term correction through clonal expansion
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