Establishment of stable cells by induction of cDNAs of functional proteins and preparation of models for evaluation of drug efficacy for creation of new drugs

H.A.Lester报道，通过将cDNA导入已知的细胞系来表达受体、酶和离子通道等重要蛋白质的细胞模型的发展，有助于建立有用的系统来评估药物疗效和创造新药。也就是说，建立功能蛋白不是内源性而是过度表达的稳定的细胞系是有用和重要的。最近，我们研究了原代培养中CaM激酶II的激活和自磷酸化，并建立了对细胞外刺激如神经递质、细胞生长因子等做出反应的细胞系。我们报道了LTP诱导过程中大鼠海马区CA1区CaM激酶II的激活。1)以合成的引物为模板，采用聚合酶链式反应的方法，获得了CaM激酶IIa、β、γ、Delta亚基的cDNAs。将其插入到表达载体pCAGGSneo中。用电泳法或脂质体转染法将其导入PC12细胞或NG108-15细胞。使用的是模拟细胞，其中只插入载体而不插入CaM激酶II亚单位亚基亚基的cDNA。2)获得稳定表达CaM激酶IIα基因的PC12细胞。3)稳定细胞系中CaM激酶IIα的表达抑制了二丁酰cAMP诱导的轴突生长。4)用免疫兔制备了CaM激酶IIDelta的特异性抗体。5)用聚合酶链式反应(PCR)方法扩增出CaM激酶II Delta亚基亚基异构体的cDNA，并进行了序列测定。CaM激酶II Delta的三种异构体均有核定位信号。6)大鼠小脑矢状面切片免疫组织化学结果显示，CaM-KⅡDelta定位于颗粒细胞的胞核，而不位于浦肯野细胞的胞核。

H.A.Lester reported that the development of cellular models to express important proteins such as receptors, enzymes, ion channels and so on by transfecting known cell lines with the cDNAs serves for the establishment of useful systems to evaluate drug efficacy and create new drugs. In other words, the establishment of stable cell lines in which functional proteins are not endogenously present, but overexpressed be useful and important.Recently, we have studied on activation and autophosphorylation of CaM kinase II in primary cultures and established cell lines in response to extracellular stimuli such as neurotransmitters, cell growth factors and so on. We reported the activation of CaM kinase II in the CA1 area of the hippocampus during LTP induction.The present study were carried out as follows. 1)We prepared the cDNAs of CaM kinase IIalpha, beta, gamma, delta subunits by the PCR method with synthesized primers. The cDNAs were inserted into the expression vector pCAGGSneo. PC12 cells or NG108-15 cells were transfected with the cDNAs by electropolation or the lipofectamine method. Mock cells into which only the vector is inserted without the cDNAs of CaM kinase II subunit isoforms were used. 2)Stable PC12 cells transfected with the cDNAs of CaM kinase IIalpha were prepared. 3)The expression of CaM kinase IIalpha in stable cell lines inhibited neurite outgrowth induced by dibutyryl cAMP.4)The specific antibody to CaM kinase IIdelta was prepared by immunizing rabbits. 5)The cDNAs of CaM kinase IIdelta subunit isoforms were prepared by the PCR method and sequenced. Three isoforms of CaM kinase IIdelta had nuclear localization signal. 6)The immunohistochemistry of the sagittal sections of rat cerebellum demonstrated that CaM kinase IIdelta is localized in the nuclei of granule cells, but not in the nuclei of Purkinje cells.