| Project/Area Number |
12556049
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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 | 展開研究 |
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| Research Field |
Basic veterinary science/Basic zootechnical science
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| Research Institution | Nagoya University (2002)
Tokyo Medical and Dental University (2000-2001) |
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Principal Investigator |
KAWAGUCHI Yasushi Nagoya University, Graduate School of Medicine, Associate Professor, 大学院・医学系研究科, 助教授 (60292984)
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| Co-Investigator(Kenkyū-buntansha) |
MAEDA Ken Yamaguchi University, Faculty of Agriculture, Associate Professor, 農学部, 助教授 (90284273)
HORIMOTO Taisuke The University of Tokyo, The institute of mideical science, Associate Professor, 医科学研究所, 助教授 (00222282)
MIKAMI Takeshi Nippon University College of Biosource Science, Professor, 生物資源科学部, 教授 (20091506)
TANAKA Michiko National institute for infectious diseases, Researcher, 感染病理部, 研究員 (10356248)
TOHYA Yukinobu The university of Tokyo, Graduate School of Agricultural and life science, Associate Professor, 大学院・農学生命科学研究科, 助教授 (20180119)
坂口 正士 (財)化学及び血清療法研究所, 第2研究部, 上級研究員
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥8,100,000 (Direct Cost: ¥8,100,000)
Fiscal Year 2002: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2001: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2000: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Keywords | herpesviruses / reverse genetics / BAC system / 組み替え法 / BAC system |
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| Research Abstract |
In recent years, several laboratories have reported on the cloning of herpesvirus genomes as bacterial artificial chromosomes (BACs) in E. coli and on procedures to manipulate these genomes using the bacterial recombination machinery. However, the herpesvirus-BACs reported so far are either replication-incompetent or infectious with a deletion of one or more viral genes due to the BAC vector insertion. As a multi-purpose clone for use in the research of herpesviruses, we attempted to generate infectious herpes simplex virus (HSV) -BACs containing the full genome of HSV-1 without any loss of^viral genes. Our results were as follows. (i)E. coli (YEbac102) harboring the full-length HSV-1 genome (pYEbac102) in which a BAG, flanked by loxP sites were inserted into the intergenic region between U_L3 and U_L4 was constructed, (ii) pYEbac102 was an infectious molecular clone, given that its transfection into rabbit skin cells resulted in production of infectious virus (YK304). (iii)' The BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus YK304 by co-infection of Vero cells with YK304 and a recombinant adenovirus AxCANCre expressing Cre recombinase. (iv) As far as was examined, the reconstituted viruses from pYEbac102 could not be phenotypically differentiated from wild-type viruses in vitro and in vivo. Thus the viruses grew as well in Vero cells as did the wild-type virus and exhibited wild-type virulency in mice on intracerebral inoculation. (v) The infectious molecular clone pYEbac102 is in fact useful for mutagenesis of the HSV-1 genome by bacterial genetics and a recombinant virus carrying amino acid substitutions in both copies of the αO gene was generated. pYEbac102 will be multi-applicable to the rapid generation of genetically engineered HSV-1 recombinants in basic research into HSV-1 and in the development of HSV vectors in human therapy.
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