| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2000: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1999: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1998: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
Ca^<2+>/calmodulin-dependent protein kinase II (calmodulin kinase II, CaMKII) is a multifunctional protein kinase which exists most abundantly in the central nervous system. CaMKII is thought to be deeply involved in the regulation of neuronal activity and synaptic plasticity. The main purpose of this study is to generate genetically engineered mice that express functionally deficient CaMKII α subunit, the major subunit of CaMKII in the forebrain, by knock-in strategy, and to obtain further insights into the biological functions of CaMKII by analyzing these mice. By using homologous recombination technique, a point mutation was introduced into the normal CaMKII α subunit gene, i.e., the amino acid residue Lys-42, which is essential for the binding of ATP, was replaced by Arg-42 in CaMKII α subunit. This altered molecule, α CaMKII (Arg-42), has no catalytic activity, but still can bind Ca^<2+>/calmodulin and form multimeric structure through association domains.
Mouse genomic DNA fragmen
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t of CaMKII α subunit was obtained from TT2 genomic DNA library by screening with rat CaMKII α subunit cDNA.After cloning into a plasmid vector, oligonucleotide-directed mutagenesis of Lys-42 to Arg-42 was accomplished by PCR and was confirmed by nucleotide sequencing. A lox P-flanked neomycine-resistance cassette and a diphtheria toxin A fragment cassette were cloned into the targeting vector. After linearization, the targeting construct was introduced into TT2-derived ES cells by electroporation. After screening with the antibiotic G418, 3 out of 410 ES clones were detected to be positive for homologous recombination by PCR and Southern blot analyses. These positive clones are now being used to generate chimeric male mice by microinjection into eight-cell embryos.
Once chimeric males are obtained, they will be bred to ICR females to identify germ line transmitters. Heterozygotes will be interbred to generate homozygous α CaMKII (Arg-42) mutant mice. These mutant mice will be useful for the functional analyses of CaMKII in vivo. Less
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