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J. Biol. Chem., Vol. 281, Issue 36, 26615-26623, September 8, 2006

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Human Artificial Chromosomes Constructed Using the Bottom-up Strategy Are Stably Maintained in Mitosis and Efficiently Transmissible to Progeny Mice^*

Nobutaka Suzuki, Kazuhiro Nishii, Tuneko Okazaki, and Masashi Ikeno¹

From the Institute for Comprehensive Medical Science, Fujita Health University and the Education and Research Center of Animal Models for Human Diseases, Fujita Health University, Toyoake, Aichi 470-1192, Japan

Human artificial chromosomes (HACs) are alternative vectors that promise to overcome problematic transgene expression often occurring with conventional vectors in mammalian cells and bodies. We have successfully generated HACs by multimerization of a cloned long alphoid stretch in a human cell line, HT1080. Furthermore, we developed technologies for cloning large genomic regions into HACs by means of co-transfection of clones with the alphoid array and clones encoding the genomic region of interest. The purpose of this study was to investigate the mitotic and meiotic stability of such HACs in mouse cells and bodies. We transferred a circular HAC containing the guanosine triphosphate cyclohydolase I gene (GCH1-HAC) and a linear HAC containing the human globin gene cluster (globin-HAC) from HT1080 cells into mouse embryonic stem (ES) cells by microcell-mediated chromosome transfer. The HACs were stably maintained in mouse ES cells for 3 months. GCH1-HACs in every ES cell line and globin-HACs in most ES cell lines maintained their structures without detectable rearrangement or acquisition of mouse genomic DNA except one globin-HAC in an ES cell line rearranged and acquired mouse-type centromeric sequences and long telomeres. Creation of chimeric mice using ES cells containing HAC and subsequent crossing showed that both the globin-HAC that had rearranged and acquired mouse type centromeric sequences/long telomeres and GCH1-HACs were retained in tissues of mice and transmitted to progeny. These results indicate that human artificial chromosomes constructed using the bottom-up strategy based on alphoid DNA are stable in mouse bodies and are transmissible.

Received for publication, March 30, 2006 , and in revised form, July 11, 2006.

^* This work was supported by a special grant for Core Research for Evolutional Science and Technology from the Japan Science and Technology Corp., a grant for establishment of transgenic agro-biofarm systems from the Ministry of Agriculture, Forestry, and Fisheries of Japan, and a grant-in-aid for Scientific Research from The New Energy and Industrial Technology Development Organization, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Institute for Comprehensive Medical Science, Fujita Health University, 1-98 Kutsukake-cho, Toyoake, Aichi, Japan 470-1192. Tel.: 81-562-93-9376; Fax: 81-562-93-8833; E-mail; mikeno{at}fujita-hu.ac.jp

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