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Short Report
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| Reduction of MBS85 gene expression after the targeted integration of a transgene into the AAVS1 site using adeno-associated virus integration machinery |
| Ajalli Rahim1, 2, Masashi Urabe1, Hiroaki Mizukami1, Akihiro Kume1 Keiichi Ichimura2 Keiya Ozawa1, 3 |
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1Division of Genetic Therapeutics, Jichi Medical University, Tochigi, Japan.
2Department of Otolaryngology-Head and Neck Surgery, Jichi Medical University, Tochigi, Japan. 3Division of Hematology, Jichi Medical University, Tochigi, Japan. |
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Article ID:100001IJGGTAR2011 doi:10.5348/ijggt-2011-1-SR-1 |
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Address correspondence to: Keiya Ozawa Division of Genetic Therapeutics, Jichi Medical University 3311-1 Yakushiji, Shimotsuke Tochigi Japan Phone: +81-285-58-7402 Fax: +81-285-44-8675 Email: kozawa@jichi.ac.jp |
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| How to cite this article: |
| Rahim A, Urabe M, Mizukami H, Kume A, Ichimura K, Ozawa K. Reduction of MBS85 gene expression after the targeted integration of a transgene into the AAVS1 site using adeno-associated virus integration machinery. International Journal of Genetics and Gene Therapy 2011;1:1-7. |
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Abstract
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Aims:
The adeno-associated 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). Using the AAV integration machinery, we performed the AAVS1-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 AAVS1-targeted insertion of the transgene on the host cells.
Methods: We co-transfected 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 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 AAVS1 affected MBS85 expression, it did not appear to cause phenotypic changes in the KM-102 cells. Conclusions: The AAVS1 site is a safe harbor for transgene insertion although it results in impaired MBS85 expression in KM-102 cells. | |
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Key Words:
Site-specific transgene integration; Adeno-associated virus; Rep protein; AAVS1 locus; MBS85
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Author Contributions:
Ajalli Rahim - Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting the article, Final approval of the version to be published Masashi Urabe - Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting and critical revision of the article, Final approval of the version to be published Hiroaki Mizukami - Conception and design, Analysis and interpretation of data, Critical revision of the article, Final approval of the version to be published Akihiro Kume - Conception and design, Analysis and interpretation of data, Critical revision of the article, Final approval of the version to be published Keiichi Ichimura - Conception and design, Critical revision of the article, Final approval of the version to be published Keiya Ozawa - Conception and design, Analysis and interpretation of data, Critical revision of the article, Final approval of the version to be published |
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Guarantor of submission:
The corresponding author is the guarantor of submission. |
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Source of support:
None |
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Conflict of interest:
The authors declare that they have no competing interests. |
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Copyright:
© Keiya Ozawa et al. 2011; This article is distributed under the terms of Creative Commons attribution 3.0 License which permits unrestricted use, distribution and reproduction in any means provided the original authors and original publisher are properly credited. (Please see www.ijcasereportsandimages.com /copyright-policy.php for more information.) et al. 2011; This article is distributed the terms of Creative Commons attribution 3.0 License which permits unrestricted use, distribution and reproduction in any means provided the original authors and original publisher are properly credited. (Please see Copyright Policy for more information.) |
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