Basic Researches on Cell Fusion and Their Application to Cell Technology

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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