Transferrinfection. A Highly Efficient Way to Express Gene Constructs in Eukaryotic Cells

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Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary cells in the presence of adenovirus

Endotoxin (lipopolysaccharide, LPS) is commonly found as a contaminant in plasmid DNA preparations. We demonstrate here that the quantities of LPS typically contaminating DNA preparations can generate a toxicity to primary cells (primary human skin fibroblasts, primary human melanoma cells) in the presence of entry-competent adenovirus particles. Toxicity can be observed with as little as 100 ng/ml free LPS or 100 pg/ml LPS when the LPS is assembled into polylysine/adenovirus complexes. Simple and effective methods of removing the contaminating LPS using either a polymyxin B resin or Triton X-114 extraction are described. Treatment of DNA samples to remove LPS eliminates the toxicity to primary cells

Un aspect de l'urbanisation en Côte d'Ivoire

Cotten Anne-Marie. Un aspect de l'urbanisation en Côte-d'Ivoire. In: Cahiers d'outre-mer. N° 106 - 27e année, Avril-juin 1974. pp. 183-193

DNA-binding transferrin conjugates as functional gene-delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety

We have previously demonstrated that transferrin-polycation conjugates are efficient carrier molecules for the introduction of genes into eucariotic cells. We describe here a more specific method for conjugation of transferrin with DNA-binding compounds involving attachment at the transferrin carbohydrate moiety. We used the polycation poly(L-lysine) or the DNA intercalator, ethidium homodimer as DNAbinding domains. Successful transferrin-receptor-mediatedd elivery and expression of the Photinus pyralis luciferase gene in K562 cells has been shown with these new transferrin conjugates. The activity of the transferrin-ethidium homodimer (TfEtD) conjugates is low relative to transferrin-polylysine conjugates; probably because of incomplete condensation of the DNA. However, DNA delivery with TfEtD is drastically improved when ternary complexes of the DNA with TfEtD and the DNA condensing agent polylysine are prepared. The gene delivery with the carbohydrate-linked transferrin-polylysine conjugates is equal or superior to described conjugates containing disulfide linkage. The new ligation method facilitates the synthesis of large quantities (>lo0 mg) of conjugates. INTRODUCTION Transferrin-polycation conjugates are efficient carriers for the uptake of DNA into eucariotic cells (I). This gene transfer technique, termed tramferrinfection, is based on receptor-mediated endocytosis of DNA complexed with polycation-transferrin conjugates (2,3). Our initial conjugate synthesis (1) involved the modification of one to two amino groups on the transferrin molecule with the bifunctional reagent succinimidyl34 2-pyridy1dithio)propionate (SPDP), followed by ligation to similarly modified polycations (polylysine or protamine) through the formation of disulfide bonds. Because there are more than 50 lysines on the large (about 80 kDa) transferrin protein, the actual site (or sites) of ligation to the polycation is unknown with this method. In this paper we describe the synthesis of new transferrin conjugates that are ligated with DNA-binding compounds in a specific manner through modification of the transferrin carbohydrate moiety. The conjugates thus obtained are free of any groups derived from chemical linking agents, since the connecting atoms are already present within the starting compounds. The carbohydrate group acts as anatural spacer that puts a 32-atom distance between the transferrin and the DNA binding moiety. This spacer effect may be important for appropriate presentation of the ligand to its receptor. As a DNA-binding compound, the polycation polylysine was used, similar to the use described in ref 1 or to the asialo-orosomucoid conjugates prepared by Wu and Wu (4). We have also prepared a novel type of transferrin conjugate that contains the DNA intercalator ethidium homodimer (5) as the DNAbinding group and demonstrate successful receptormediated gene delivery with these conjugates. EXPERIMENTAL PROCEDURES Human transferrin (iron-free), conalbumin (iron-free), and poly(L-lysine) were obtained from Sigma. Liquid chro- Abbreviations used: FITC, fluorescein ieothiocyenate; TfEtD, traneferrin-ethidium homodimer conjugate; TfpL, traneferrinpolytL- lysine) conjugate; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid

An examination of nonverbal communication in the business community

LD2668 .R4 SPCH 1989 C68Master of ArtsCommunication Studie

Transferrin-polycation conjugates as carriers for DNA uptake into cells.

We have developed a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells. We accomplished this by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, as well as the chicken homologue conalbumin, has been covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with double-stranded DNA, single-stranded DNA, and modified RNA molecules independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs). When complexes of transferrin-polycation and a bacterial plasmid DNA containing the gene for Photinus pyralis luciferase are supplied to eukaryotic cells, high-level expression of the luciferase gene occurs, demonstrating transferrin receptor-mediated endocytosis and expression of the imported DNA. We refer to this delivery system as "transferrinfection.