| Project/Area Number |
04454170
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Osaka University |
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Principal Investigator |
SHIMADA Kazunori Osaka Univ.Res.Inst.for Microb.Dis.Professor, 微生物病研究所, 教授 (40037354)
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| Co-Investigator(Kenkyū-buntansha) |
NISHIGUCHI Seiji Osaka Univ.Res.Inst.for Microb.Dis.Instructo, 微生物病研究所, 助手 (90237686)
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| Project Period (FY) |
1992 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1993: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1992: ¥4,500,000 (Direct Cost: ¥4,500,000)
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| Keywords | Targeted integration / Gene targeting / Homologous recombination / ES cell / F9 cell / Transthyretin. / Methylation / Inherited disease / モデルマウス / 選択マーカー / 遺伝子標的組込み |
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| Research Abstract |
We developed two kinds of gene targeting vectors using mouse embryonal carcinoma F9 cell and a transthyretin (ttr) gene as a model system. We used F9 cell, because it is easy to handle and has sinilar properties to embryonal stem cell, and used the ttr gene, because mutations of the human gene cause familial amyloidotic polyneuropathy.
1. A vector to introduce insertional mutations : This vector consisted of a 5.9 kd ttr fragment carrying neomycin-resistance (neo) gene in the 2nd exon and a HSV-tk (herpes simplex virus thymidine kinase) gene at the 3' end. When the plasmid DNA tail at the 3' end of HSV-tk gene was longer, the efficiency of GANC selection was higher. Structues of the vector DNAs retained in non-targeted clones suggested their exonucleolytic degradation. Capping the ends of vector DNAs with hairpin-shaped oligonucleotides increased the efficiency of GANC selection.
2. A vector to introduce subtle mutations : This vector consisted of the ttr fragment carrying a 3-base mutation in the 2nd exon that causes a single amino acid substitution in TTR identical to FAP patients, and a cassette of neo and HSV-tk genes flanked with a 3 kd duplication of the 2nd intron of ttr gene. In the 1st step, those clones, in which part of the endogenous ttr gene was replaced by vector DNA through homologous recombination, were selected from G418^r clones. In the 2nd step, GANC^r clones, in which the selection marker cassette had been excised by intrachromosomal recombination, were selected from the targeted G418^r clones. Interestingly, many GANC^r clones carried HSV-tk gene methylated at the promoter region. The selection marker cassette was efficiently excised in 5 random integrants of the vector DNA,indicating that this step is applicable to a wide variety of mouse genes.
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