RESUMEN
With the goal of developing non-viral techniques for exogenous gene delivery into mammalian cells, we have studied receptor-mediated gene transfer using complexes of plasmid DNA and galactosylated poly-L-lysine, poly(L-Lys)Gal. To evaluate the optimal parameters for efficient gene transfer into human hepatoma HepG2 cells by the DNA-poly(L-Lys)Gal complexes, the bacterial reporter genes lacZ and cat were used. Examination of the reporter gene expression level showed that the efficiency of DNA delivery into the cells depends on the structure of DNA--poly(L-Lys)Gal complexes formed at various ionic strength values. The efficiency of DNA transfer into the cells also depends on DNA/poly(L-Lys)Gal molar ratio in the complexes. Plasmid vector carrying human apolipoprotein A-I (apoA-I) gene was injected as its complex with poly(L-Lys)Gal into rat tail vein. Some level of ApoA-I was detected in the serum of the injected rats. Also, the human apoA-I-containing plasmid was found to be captured specifically by the rat liver cells and transported into the cell nuclei, where it can persist as an episome-like structure for at least a week. After repeated injections of DNA--poly(L-Lys)Gal complexes, the level of human ApoA-I in rat serum increases, probably, due to accumulation of functional human apoA-I gene in the liver cell nuclei. The data seem to be useful for the development of non-viral approaches to gene therapy of cardiovascular diseases.
Asunto(s)
ADN/administración & dosificación , Galactosa/metabolismo , Polilisina/administración & dosificación , Células 3T3 , Animales , Apolipoproteína A-I/genética , Arteriosclerosis/genética , Arteriosclerosis/terapia , Cloranfenicol O-Acetiltransferasa/genética , Genes Reporteros , Terapia Genética , Humanos , Técnicas In Vitro , Operón Lac , Hígado/metabolismo , Ratones , Polilisina/metabolismo , Ratas , Células Tumorales CultivadasAsunto(s)
Regulación Enzimológica de la Expresión Génica , Glucuronidasa/genética , Lisosomas/enzimología , Testosterona/fisiología , Animales , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucuronidasa/metabolismo , Riñón/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Orquiectomía , Especificidad de Órganos , Pene/enzimología , Glándulas Salivales/enzimología , Testosterona/farmacologíaRESUMEN
To study structure-function relationships in low density lipoprotein receptor (LDLR), a key protein in human cholesterol metabolism, it is reasonable to operate with separate protein domains. To obtain highly purified functionally active LDLR ligand-binding domain, we have cloned the corresponding LDLR cDNA fragment in two expression plasmid vectors of Escherichia coli. We have developed methods to purify fusion and practically individual recombinant proteins and characterized the obtained products biochemically. Antibodies raised against fused with beta-galactosidase and individual recombinant protein have been shown to be efficient in identification of LDLR protein in crude lysates of human fibroblasts (cell line HT-1080).