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Reduction of MBS85 Gene Expression after the Targeted Integration of a Transgene Into the AAVS_1 Site Using Adeno-Associated Virus Integration Machinery
並列摘要
Introduction: The adeno-associated virus (AAV) integrates into a particular location on human chromosome 19 (19q13.4), which is known as AAVS_1, through the binding of a replication protein, Rep, to the viral inverted terminal repeat (ITR). Aims: Using the AAV integration machinery, we performed the AAVS_1-targeted insertion of a reporter gene into KM-102 cells, a stromal cell line established from human bone marrow cells, and examined the impact of the AAVS_1-targeted insertion of the transgene on the host cells. Methods: We co-transfected KM102 cells with a Rep plasmid and a plasmid containing GFP and a blasticidin resistance gene flanked by ITR sequences. After culturing the cells with blasticidin S, thirty clones were obtained and analyzed for AAVS_1-specific integration and then had their myosin binding subunit 85 (MBS85) mRNA levels measured. Results: Out of 30 selected clones, three clones containing the GFP gene at AAVS_1 were obtained. These three clones grew well, similar to the wild-type KM-102 cells, but showed a decreased level of MBS85 mRNA expression. These results indicated that although the insertion of the transgene at AAVS_1 affected MBS85 expression, it did not appear to cause phenotypic changes in the KM-102 cells. Conclusions: The AAVS_1 site is a safe harbor for transgene insertion although it results in impaired MBS85 expression in KM-102 cells.