| Project/Area Number |
16500254
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurochemistry/Neuropharmacology
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| Research Institution | Tokyo Metropolitan Organization for Medical Research |
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Principal Investigator |
NUKADA Toshihide Tokyo Metropolitan Organization for Medical Research, Tokyo Institute of Psychiatry, Head, 東京都精神医学総合研究所, 副参事研究員 (80189349)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2004: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | P / Q-type Ca^<2+> channel / α1A subunit / Protein kinase A / Knock-in mouse / Bacterial homologous recombination / loxP / Cre recombinase / ES cell / マウスゲノム / Creリコンビナーゼ / loxP |
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| Research Abstract |
For studies on the physiological roles of phosphorylation of P/Q-type Ca^<2+> channels in vivo, we generated knock-in mice with substitution of a critical threonine residue on the α1A subunit (denoted as Thr-PKA) in the modulation of channels by cyclic AMP-dependent protein kinase (PKA). 1.For targeting constructs, a 16 kb genomic α1A DNA containing the exon that coded for Thr-PKA (denoted as exon-Thr^*) was cloned from mouse genomic library. 2.By using bacterial homologous recombination procedures, we constructed a targeting vector composed of a substitution of Thr-PKA with Ala and an insertion of loxP-STOP (a transcriptional/translational stop cassette)-NEO (a neo transcription unit)-loxP in the upstream intron from exon-Thr^*, as well as an addition of herpes tk transcription unit at the 5' end of the genomic α1A DNA. In this construct, the C-terminus of the α1A subunit was truncated by the function of STOP cassette. After removal of STOP-NEO by the site-specific recombination with Cre, the point mutation of Thr-PKA was achieved in the α1A gene. 3.Next, this targeting vector was introduced into mouse ES cells with electroporation, and 15 ES cells that had correctly incorporated the targeting construct into the genome were isolated from 250 cells. 4.In the gene-targeted ES cells, the STOP-NEO sequence was removed from the genome by Cre-mediated recombination with an efficiency of 10%. 5.Knock-in mice were generated by microinjecting these gene-targeted mouse ES cells into mouse blastocysts. The injected blastocysts were then transferred to pseudopregnant females and allowed to develop to term.
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