1996 Fiscal Year Final Research Report Summary
The establishment of prion protein humanaized mice using homologous recombination
| Project/Area Number |
06404032
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurology
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| Research Institution | Tohoku University (1996)
Kyushu University (1994-1995) |
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Principal Investigator |
KITAMOTO Tetsuyuki Schoo, of Medicine, Tohoku University, professor, 医学部, 教授 (20192560)
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| Co-Investigator(Kenkyū-buntansha) |
NAKAMURA Kenji Kyusyu University, assistant professor, 生体防御研究所, 助手 (90253533)
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| Project Period (FY) |
1994 – 1996
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| Keywords | prion protein / homologous recombination / site-specific recombination / humanized mice / transgenic mice |
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| Research Abstract |
To study human prions, we previously used the mice with wild type murine PrP gene for the transmission experiment. Mice have been used to do the transmission experiments from human or other animals with prion diseases. However, it takes long time for the successful transmission with the wild type mice, and the successful transmission was rarely observed in the wild type mice. Therefore, it is essential to establish humanized prion protein mice for the transmission experiment from human prion diseases. For this purpose, we designed the gene replacement method with the homologous recombination. Our method was based on the site-directed recombination using Cre-loxp system. At first, the loxp-neo-gpt-loxp cassette was used for the homologous recombination. One clone out of 140 G418-resistant clones was selected by the Southern blotting. We made the chimeta mice from this clone, and found out the germinal transmission from the chimera mice. The Cre plasmid was micro-injected in the fertiliz
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ed eggs from the heterozygous mice.
In 6 out 7 mice, the successful site-specific recombination was observed. We established the humanized mice using the homologous recombination and the site-specific recombination. In addition, we used the loxp-neo-loxp cassette for making other types of humanized mice. With this cassette, the efficient homologous recombination was obtained. One out of 60 clones or 3 out of 120 clones were recognized positively by the Southern blot analysis. At present, we are making the chimera mice from these ES cells.
For establishment of mice overexpressing human prion protein, we also made the transgenic mice. The natural promoter and the natural gene structure was obtained from the genomic library of the 129sv or I/ln mice. The transgene was constructed with the natural murine prion protein gene except for the exon 3. The exon 3 was replaced with human prion protein. Two constructs was micro-injected in the fertilized eggs from C57bl mice. We obtained 5 or 4 transgenic founder mice from each construct. These mice or germ-lined F1 mice showed a high expression of human prion protein mRNA.These replacement or transgenic mice were useful to analyze the human prion titer. Less
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