Introduction of Artifitial Translocator into Plasma membrane in Cultured Human Cell-Line

细胞被生物膜包围，生物膜通过特定的转运体渗透到特定的底物中。因此，即使是培养的细胞，也很难通过更换额外的培养液来控制细胞条件。我们试图通过两种方法来控制细胞的能量状态。首先，我们尝试分离氧化磷酸化缺陷的突变细胞系。其次，我们试图通过引入操控基因来整合人工转位蛋白；在这种情况下，我们使用了ADP/ATP转位蛋白和逆转录病毒囊膜蛋白的融合蛋白，该蛋白针对质膜。1)利用从嗜热菌中提纯的热稳定性质子转运ATPase制作了ATP传感器。2)从线粒体肌病患者体内建立了细胞色素C氧化酶缺陷细胞系。3)从1例丙酮酸脱氢酶缺陷症患者出发，建立丙酮酸脱氢酶缺陷型细胞株。4)用视黄醇处理人早幼粒白血病细胞HL-60，获得线粒体缺陷细胞。5)克隆并测序人ADP/ATP转运蛋白基因。6)在SV40启动子的作用下，将ADP/ATP转运蛋白基因与逆转录病毒囊膜基因融合。将构建的基因导入小鼠成纤维细胞。得到的转化子不稳定，不能维持。因此，将新霉素抗性基因直接连接到HeLa细胞中，并将其导入HeLa细胞。实验正在进行中。

A cell is surrounded by biomembranes, which permeate specific substrates via specific translocators. Therefore, even if the cultured cell, it is hard to control the cellular conditions by changing the extra culture medium. We have attempted to control the cellular energy state by two methods. First, we have tried to isolate the mutant cell lines which are deficient in oxidative phosphorylation. Second, we have tried to incorporate an artificial tanslocator by introducing the manipulated gene; in this case, we use a fusion protein of ADP/ATP translocator with a retro virus envelop protein, which target plasma membrane. The following results were obtained by several trials.1) To detect the ATP concentration easily, an ATP sensor was made by using thermo-stable proton translocating ATPase purified from a thermophilic bacterium.2) The cytochrome C oxidase deficient cell line were established from a patient with mitochondrial myopathy. The cell survive could be controlled by substrate for oxidative phospholylation.3) The pyruvate dehydrogenase deficient cell line was established from a patient with its deficiency. The defective gene were analyzed in detail.4) The mitochondrial deficient cell were made by treating the human promyelocytic leukemia cell HL-60 with retinol. In addition, the gene expression of mitochondrial proteins were also examined by Northern blotting method.5) The human cDNA encoding ADP/ATP translocator was cloned and sequenced.6) The ADP/ATP translocator cDNA was fused to a retro virus envelop gene under SV40 promoter. The constructed gene was transfected into mouse fibroblast cells. The resulting transformant was unstable and could not be maintained. Therefore, neomycin resistant gene was connected directly and then transfected to HeLa cells. The experiment is just on the way.