Studies on the coupling of DNA to low density lipoproteins (LDL) and the interaction of these complexes with eukaryotic cells.

Abstract

The application of Molecular Biochemistry for transfection studies in eukaryotic systems is well documented. Of the numerous methods employed for the introduction of foreign DNA into eukaryotic cells, the use of low density lipoproteins (LDL) as carriers of DNA into cells has not been reported. LDL was isolated, characterized with respect to its protein and lipid components, and then variously modified in an attempt to enhance its affinity for DNA. It was found that both unmodified and modified LDL could interact with DNA, at physiological pH. The carbodiimide modified LDL (ECDI - LDL) showed the greatest affinity for DNA. LDL and ECDI - LDL were used to study LDL receptor binding in skin fibroblasts. This was followed by a study of receptor binding activities of both unmodified LDL and ECDI - LDL complexed to DNA (pBR322). Although the extent of binding of ECDI - LDL and ECDI - LDL - DNA complexes to plasma membranes was greater, the internalization and degradation of both modified and unmodified LDL complexes were equivalent. This additional binding was attributed to non - receptor - specific affinity of the carbodiimide modified complexes for the plasma membrane. The transfection of foreign DNA into eukaryotic cells in culture was monitored by assaying for the expression of the cloning vector, pSV2cat, complexed to LDL or ECDI - LDL and introduced into the cells by LDL receptor - mediated endocytosis. Of the cell lines in which the expression of the pSV2cat recombinant DNA was monitored, the human lung fibroblasts showed the greatest activity of the expressed chloramphenicol acetyl transferase enzyme. Although transfection efficiency was lower than that of the calcium phosphate - DNA coprecipitation procedure, the LDL receptor - mediated transfection of eukaryotic cells was carried out under physiological conditions and may be applicable in vivo.