ABSTRACT
Naked plasmid DNA (pDNA) injection could become an alternative procedure to viral and nonviral gene delivery systems. We have previously shown that Epstein-Barr virus (EBV)-based plasmid vectors containing the EBV nuclear antigen 1 (EBNA1) gene and the oriP sequence enable quite high and long-lasting expression in various in vitro and in vivo transfection systems. The EBV-based plasmids were intravenously injected into mice via their tail vein under high pressure. A large amount of the marker gene product was expressed in the liver; as much as 320 microg of luciferase was demonstrated per gram of liver at 8 to 24 h after a single injection with 10 microg of DNA. More than 70% of liver cells stained with X-gal when beta-gal gene was transferred. The expression level was significantly higher than that obtained by conventional pDNA lacking the EBNA1 gene and oriP. On day 35 after the transfection, the expression from the EBV-based plasmid was approximately 100-fold stronger than the conventional pDNA gene expression. Both the EBNA1 gene and oriP are a prerequisite for the augmentation of the transfection efficiency. These results suggest that the intravascular transfection with naked EBV-based plasmid may provide a quite efficient, simple and convenient means to transduce therapeutic genes in vivo into the liver.
Subject(s)
Genes, Viral , Genetic Therapy/methods , Herpesvirus 4, Human/genetics , Liver/metabolism , Transfection/methods , Vaccines, DNA/administration & dosage , Animals , Female , Gene Expression , Injections, Intravenous , Luciferases/genetics , Mice , Mice, Inbred BALB C , beta-Galactosidase/geneticsABSTRACT
Thylakoid membranes and Photosystem I (PS I) complexes were isolated from a glaucocystophyte, Cyanophora paradoxa, which is thought to have the most primitive 'plastids', and the proteins related to PS I were examined. The intrinsic light-harvesting chlorophyll protein complexes of PS I (LHC I) were not detected by an immunological method. The PS I complexes consisted of at least eight low-molecular-mass proteins in addition to PS I reaction center proteins. The N-terminal sequence of the PsaD protein has higher homology to that of Chlamydomonas reinhardtii and land plants, than to that of other algae or cyanobacteria. On the other hand, the PsaL sequence has the highest homology to those of cyanobacteria. Taking into account the other sequences of PS I components whose genes are encoded in the cyanelle genome, and the fact that LHC I is not detected, it is concluded that PS I of C. paradoxa has chimeric characteristics of both 'green' lineages and cyanobacteria.
ABSTRACT
We have estimated the efficiency of Epstein-Barr virus (EBV)-based vectors in transfecting genes into cell lines of lympho-hematopoietic lineages. The transfection efficiency was estimated both at transient and stable phases, in terms of expression of a marker gene and acquisition of drug resistance, respectively. Plasmid vectors carrying EBV oriP (replication origin of plasmid), EBNA (EBV nuclear antigen)-1 and as the marker genes, murine CD8 alpha cDNA and neoR (neomycin resistant) genes were transfected into various cell lines by electroporation. When cell lines constitutively expressing EBNA-1 were transduced, virtually all the cells expressed CD8 alpha on day 3 and acquired G418 resistance thereafter. In the case of K562 cells, which do not express EBNA-1, approximately 40% of cells expressed the marker gene product on day 3 posttransfection, and 30% of cells became stable transfectants. These data suggest a broader application of the EBV vector system in basic immunology and medicine.
