Abstract

Introduction: The adeno­associated virus (AAV) integrates into a particular location on human chromosome 19 (19q13.4), which is known as AAVS1, through the binding of a replication protein, Rep, to the viral inverted terminal repeat (ITR). Aims: Using the AAV integration machinery, we performed the AAVS1­targeted insertion of a reporter gene into KM­102 cells, a stromal cell line established from human bone marrow cells, and examined the impact of the AAVS1­targeted insertion of the transgene on the host cells. Methods: We co­transfected KM­ 102 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 AAVS1 ­ 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 AAVS1 were obtained. These three clones grew well, similar to the wild­type KM­102 cells, but showed a decreased level of MBS85 mRNA expression. These results indicated that although the insertion of the transgene at AAVS1 affected MBS85 expression, it did not appear to cause phenotypic changes in the KM­102 cells. Conclusions: The AAVS1 site is a safe harbor for transgene insertion although it results in impaired MBS85 expression in KM­102 cells.

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