| Co-Investigator(Kenkyū-buntansha) |
NARITOMI Kenji University of the Ryukyus, Faculty of Medicine, Professor, 医学部, 教授 (20101446)
YANAGI Kumiko University of the Ryukyus, Faculty of Medicine, dssisfant, 医学部, 助手 (90294701)
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| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2006: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
In order to know the position effect and stability of gene expression at various regions in human artificial minichromosomes, we have constructed four types of modified minichromosomes, which was introduced a BAC harboring a whole gene unit (HPRT, or Factor IX) into various sites such as near telomere, near centromere, and euchromatic region, and we investigated intensity and stability of the gene expression.
Fist, we constructed plasmid vectors, which could introduce lox71 (mutant loxp) into the human minichromosome at various sites by homologous recombination or telomere targeting method. We also constructed a telomere-targeting vector, which could replace the neoR gene to the ZeoR gene located at the telomeric region in the minichromosome.
Then, we modified the minichromosome using the vectors above, and introduced a BAC (HPRT-66 or F9-66) into the modified minichromosomes by the Cre/mutant lox recombination system. The efficiencies of BAC introduction into the minichromoscmes were approximately 75% and 70% for HPRT-66 BAC and F9-66 BAC, respectively.
Relative gene expressions of human HPRT gene estimated by qPCR were 100, 83, and 122, when the gene was located near telomere near centromere, and euchromatic region, respectively. Copy number of the modified minichromosomes in DT40 cells was 1 copy/nucleus (approximately 70%) or 2 copies/nucleus (approximately 30%). Relative expression of the human HPRT gene was 15% of authentic Hprt gene expression in DT40. The gene expression was not significantly changed in any modified minichromosomes after 60 days culture.
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