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a Departments of Molecular and Cell Genetics,
b Human Genome Sciences (Kirin Brewery), and
c Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medicine, Tottori University, Yonago, Tottori, Japan;
d Department of Tissue Regeneration, Institute for Frontier Medical Science, Kyoto University, Sakyo-ku, Kyoto, Japan
Key Words. Human artificial chromosome vector • Insulator • Mesenchymal stem cells • Cell lineage–specific transgene expression • Microcell-mediated chromosome transfer • Differentiation
Correspondence: Mitsuo Oshimura, Ph.D., Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan. Telephone: 81-859-348261; Fax: 81-859-348134; e-mail: oshimura{at}grape.med.tottori-u.ac.jp
Mesenchymal stem cells (MSCs) hold promise for use in adult stem cell–mediated gene therapy. One of the major aims of stem cell–mediated gene therapy is to develop vectors that will allow appropriate levels of expression of therapeutic genes along differentiation under physiological regulation of the specialized cells. Human artificial chromosomes (HACs) are stably maintained as independent chromosomes in host cells and should be free from potential insertional mutagenesis problems of conventional transgenes. Therefore, HACs have been proposed as alternative implements to cell-mediated gene therapy. Previously, we constructed a novel HAC, termed 21
pq HAC, with a loxP site in which circular DNA can be reproducibly inserted by the Cre/loxP system. We here assessed the feasibility of lineage-specific transgene expression by the 21pq HAC vector using an in vitro differentiation system with an MSC cell line, hiMSCs, which has potential for osteogenic, chondrogenic, and adipogenic differentiation. An enhanced green fluorescent protein (EGFP) gene driven by a promoter for osteogenic lineage-specific osteopontin (OPN) gene was inserted onto the 21 pq HAC and then transferred into hiMSC. The expression cassette was flanked by the chicken HS4 insulators to block promoter interference from adjacent drug-resistant genes. The EGFP gene was specifically expressed in the hiMSC that differentiated into osteocytes in coordination with the transcription of endogenous OPN gene but was not expressed after adipogenic differentiation induction or in noninduction culture. These results suggest that use of the HAC vector is suitable for regulated expression of transgenes in stem cell–mediated gene therapy.
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