Basic Researches on Cell Fusion and Their Application to Cell Technology

在膜融合反应机理方面，(1)利用磷脂生物合成缺陷突变株，证实了磷脂酰丝氨酸等几种脂类对融合反应的需求。(2)胆固醇与F-糖蛋白F_1亚基N-末端结构域的特异性结合是细胞膜与膜融合反应的前提。(3)从需要低密度脂蛋白中胆固醇的哺乳动物细胞中分离到一个内切酶和溶酶体间运输缺陷的突变体。该突变体可用于分析溶酶体膜与内体膜的融合机制。(4)哺乳动物细胞的电融合需要细胞内钙离子，但胞外钙离子的去除并不妨碍该反应。与钙蛋白酶不同的是，内源性硫醇蛋白水解酶(S)参与了融合反应。(5)在HVJ诱导的上皮融合的情况下。细胞，它是在…发现的更多的是病毒从顶端攻击细胞，导致细胞在基外侧膜融合。因此，病毒的攻击部位远离膜融合部位，提示一些膜融合信号(S)可能在细胞内从病毒攻击部位传递到膜融合部位。(6)作为细胞融合在细胞操纵方面的应用，DNA修复机制缺陷--着色性干皮病模型的建立可能是首先定位的。XPAC(XPAC)基因是我们几年前分离到的。在本项目中，利用定点突变技术证明了XPAC基因产物的锌指基序是其DNA修复功能所必需的，并且该蛋白在一个碱性区域中含有核定位信号。用基因芯片技术打乱小鼠胚胎干细胞的Xpac基因，并将含有基因打乱细胞的嵌合体(S)注射到正常小鼠胚胎中，成功地获得了体细胞嵌合体。此外，我们还成功地打乱了ES细胞的XPAC基因的两条序列。这些细胞经紫外线处理后，DNA修复出现缺陷。因此，生产具有XPAC缺陷的模型鼠现在成为可能。(7)建立了从ES细胞制备生殖系嵌合体的方法。此外，几乎建立了一种将去核的正常小鼠卵子与基因破坏的ES细胞融合来生产小鼠的方法。(8)建立了含白喉毒素A亚单位脂质体治疗亚急性硬化性全脑炎的方法。较少

On the mechanism of membrane fusion reaction, (1) Requirement of some species of lipid, such as phosphatidylserine, for fusion reaction was substantiated by using mutants defective for phospholipid biosynthesis. (2) Specific binding of cholesterol to N-terminal domain of F_1-subunit of F-glycoprotein was shown to be a prerequisite for fusion reaction between cell membranes and membrane of HVJ (Sendai virus). (3) A mutant with a defect in traffic between endosome and lysosome was isolated from mammalian cells which required cholesterol in LDL for their growth. This mutant seems to be very useful for analysis of fusion mechanism of endosomal membranes with lysosomal ones. (4) For electrofusion of mammalian cells, intracellular Ca_<2+> is required, but removal of extracellular Ca_<2+> did not hamper the reaction. Endogenous thiol-protease (s) which is different from calpain was shown to participate in the fusion reaction. (5) In case of HVJ-induced fusion of epithelial. cells, it was foun … More d that attack of the cells by the virus from apical side resulted in cell fusion at batholateral membranes. Thus, the sites of attack of the virus were far from the sites of membrane fusion, suggesting that some signal (s) for membrane fusion may be transmitted within the cells from the virus-attacked sites to the membrane fusion sites.As an application of cell fusion for cell manipulation, (6) construction of model mouse for Xeroderma pigmentosum (XP), a defect of DNA repair mechanism, might be sited first. XPAC (XP group A complementing) gene was isolated by us several years ago. In this project, it was shown using site-directed mutagenesis that zinc finger motifs of the XPAC gene product are required for its DNA repair function, and that the protein contains nuclear localization signal in a basic domain. Disrupting XPAC gene of mouse ES (embryonic stem) cells by genetageting, and making chimera (s) containing the gene-disrupted cells by injecting them in normal mouse embryos, we have succeeded i. n obtaining a somatic cell chimera. Furthermore, we were also successful for disrupting both alleies of XPAC gene of ES cells. These cells showed defect in DNA repair after UV treatment. Thus, productions of model mouse with XPAC defect now become possible. (7) A method of production of germinal line chimeras from ES cells was established. Furthermore, a method of production of mouse derived from fusion of denucleated un-fertilized normal mouse eggs with gene-disrupted ES cells was almost established. (8) A curing method of SSPE (subacute sclerosing panencephalitis) using liposomes containing subunit A of diphteria toxin was established. Less