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

Ca^<2+>/calmodulin-dependent protein kinase II （calmodulin kinase II, CaMKII）是一种主要存在于中枢神经系统的多功能蛋白激酶。CaMKII被认为深度参与神经元活动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表达CaMKII前脑主要亚基CaMKII α亚基功能缺陷的基因工程小鼠，并通过对这些小鼠的分析进一步了解CaMKII的生物学功能。利用同源重组技术，在正常CaMKII α亚基基因中引入点突变，将CaMKII α亚基中ATP结合必需的氨基酸残基Lys-42替换为Arg-42。这种改变后的分子α CaMKII （Arg-42）没有催化活性，但仍然可以结合Ca^<2+>/钙调蛋白，并通过结合结构域形成多聚体结构。用大鼠CaMKII α亚基cDNA进行筛选，从TT2基因组DNA文库中获得了更多的CaMKII α亚基。将Lys-42克隆到质粒载体上后，通过PCR实现了对Arg-42的寡核苷酸定向诱变，并通过核苷酸测序进行了验证。将一个lox p -侧翼的新霉素耐药盒和一个白喉毒素A片段盒克隆到目标载体中。线性化后，通过电穿孔将靶向结构引入tt2衍生的ES细胞。经抗生素G418筛选，410个ES克隆中有3个通过PCR和Southern blot检测同源重组阳性。这些阳性克隆现在正被用于通过显微注射到8细胞胚胎中来产生嵌合雄性小鼠。一旦获得嵌合雄性，它们将与ICR雌性交配以鉴定生殖系传递者。杂合子将杂交产生纯合子α CaMKII （Arg-42）突变小鼠。这些突变小鼠将有助于CaMKII在体内的功能分析。少

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