| Project/Area Number |
05680741
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Laboratory animal science
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| Research Institution | Keio University |
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Principal Investigator |
SHIMODA Kouji Keio Univ.School of Medicine, Research Associate, 医学部, 助手 (00129470)
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| Co-Investigator(Kenkyū-buntansha) |
MAEJIMA Kazuyoshi Keio Univ.School of Med.Professor, 医学部, 教授 (70051464)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1994: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1993: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Transgenic mice / Homologous recombination / Introduction protocol |
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| Research Abstract |
Production of transgenic mice is now an important tool for the investigation of biomedical research and laboratory animal science. DNA fragments for microinjection are usually used as the minigene construct composed of promoter, coding and terminal sequences, or the genomic fragment encompassed by 5' regulatory and 3' terminal regions. Longer DNA fragment including more informations must have advantages for the precise gene expression in spatial and temporal manner to mimic the original gene expression patterns. Therefore, the method to introduce huge DNA fragments into mouse genome is required for investigation of genes and gene families which are extended for several hundred kilobase long. Our previous research indicated that the fragments injected into fertilized mouse eggs combined to constitute larger fragments by homologous recombination in the overlapping regions of their terminals. In this project we determined the nucleotide sequences in the conjunct regions of homologous and heterogenous recombination by PCR-aid cloning and sequencing. Non-overlapping fragments injected were combined in head to tail orientation, and the deletion and replacement of several nucleotides were observed in the junctions. Overlapping fragments were combined by homologous recombination in their overlapping regions and the replacement of few nucleotide was observed around the junction, however, the deletion of nucleotides was not detected. These results indicated that the reconstitution of large fragments by homologous recombination was a useful protocol for introduction of huge DNA molecules on the mouse genome.
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