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Functional analysis of Ca^<2+>/calmodulin-dependent protein kinase II using genetically engineered animals.

使用基因工程动物对Ca 2+ /钙调蛋白依赖性蛋白激酶II进行功能分析。

基本信息

批准号：
10680756
负责人：
YAMAGATA Yoko
金额：
$ 2.05万
依托单位：
Okazaki National Research Institutes
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

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 … More 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

钙调蛋白依赖性蛋白激酶II（CaMKII）是一种多功能蛋白激酶，在中枢神经系统中大量存在。CaMKII被认为深深地参与神经元活性和突触可塑性的调节。本研究的主要目的是通过敲入策略产生功能缺陷型CaMKII α亚基（前脑中CaMKII的主要亚基）的基因工程小鼠，并通过分析这些小鼠来进一步了解CaMKII的生物学功能。通过同源重组技术，将点突变引入正常CaMK II α亚基基因，即，CaMK Ⅱ α亚基中与ATP结合所必需的氨基酸残基Lys-42被Arg-42取代。这种改变的分子α CaMK Ⅱ（Arg-42）没有催化活性，但仍能结合Ca^2+/钙调素，并通过结合域形成多聚体结构。 ...更多信息以大鼠CaMK Ⅱ α亚基cDNA为模板，从TT 2基因组文库中筛选CaMK Ⅱ α亚基，克隆到质粒载体中，经PCR和测序鉴定，完成了Lys-42突变为Arg-42的突变。将lox P侧翼的新霉素抗性盒和白喉毒素A片段盒克隆到靶向载体中。线性化后，通过电穿孔将靶向构建体引入TT 2衍生的ES细胞中。经抗生素G418筛选后，PCR和Southern杂交分析，从410个ES克隆中检测到3个同源重组阳性。这些阳性克隆现在正被用于通过显微注射到八细胞胚胎中来产生嵌合雄性小鼠。一旦获得嵌合雄性小鼠，它们将与ICR雌性小鼠交配，以确定生殖系传递者。将杂合子杂交以产生纯合α CaMKII（Arg-42）突变小鼠。这些突变小鼠将有助于体内CaMK Ⅱ的功能分析。少