2000 Fiscal Year Final Research Report Summary
Gene transfer methods using mammalian artificial chromosomes
| Project/Area Number |
10044326
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | Fujita Health University |
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Principal Investigator |
OKAZAKI Tuneko (Institute for comprehensive medical science, Fujita Health University Professor), 総合医科学研究所, 教授 (10022584)
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| Co-Investigator(Kenkyū-buntansha) |
MASASHI Ikeno Fujita health university, Research Associate, 総合医科学研究所, 助手 (80298546)
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| Project Period (FY) |
1998 – 2000
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| Keywords | HAC / Cre / loxP / Alphoid sequence / Microcell-mediated chromosome transfer / Ad / HAC vector |
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| Research Abstract |
Our research objective is the development of basic technologies to utilize mammalian artificial chromosomes for gene delivery vectors and we collaborated with Prof. P.Hieter at Columbia University and Prof. K.Mitani at California University to proceed the research. Okazaki's group challenged the construction of HACs in which a YAC clone containing a large human genomic area was used as a starting material. The arms of the YAC clone were modified with human telomere sequences, loxP sequence and mammalian selection marker. To insert alphoid sequence into the modified YAC by Cre/loxP recombination system, a BAC carrying 100kb alphoid sequence and loxP sequence was constructed. In order to generate HACs, the method of the YAC purification free from yeast genomic DNA was crucial and we obtained useful information through discussion with Prof. Hieter. In order to use this HACs as gene delivery vectors in the other cell lines, we transferred HACs, which had been formed in the HT1080 cell line, into mouse A9 cells by microcell-mediated chromosome transfer (MMCT) method. We confirmed that HACs transferred into the A9 cells were stably maintained. Mitani's group in collaboration with our group succeeded in the construction of Ad/HAC vector containing human telomere sequences and alphoid sequence in a helper-dependent adenovirus vector. When this DNA was introduced into HT1080 cells by lipofection, transformants were obtained. We are currently analyzing the HAC formation in the transformants. It is important future subject whether HAC is formed efficiently from Ad/HAC vectors when infected to cells as virus particles.
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