Gene transfer by receptor-mediated endocytosis : stable expression of NEO following insulin-directed entry into HepG2 cells.
Huckett, Barbara Isobel.
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Evidence is presented for DNA delivery to cultured HepG2 hepatoma cells via the endocytotic pathway, under the direction of insulin, in a soluble system of transfection leading to stable gene expression. Serum albumin treated at pH 5.5 and 20°C for 48-60h with the water-soluble carbodiimide N-ethyl-N'(3-dimethylaminopropyl) carbodiimide hydrochloride has been found to produce positively charged N-acylurea albumin capable of binding different types of DNA in a reaction which is at least partially electrostatic in nature (Huckett et al, 1986). N-Acylurea albumin, synthesised at an albumin to carbodiimide mole ratio of 1 : 500, resulting in the attachment of 27 Nacylurea moieties per albumin molecule, was covalently conjugated to insulin by glutaraldehyde cross-linkage in order to produce a macromolecule, insulin-[N-acylurea albumin], with the facilities f or both DNA transport and receptor binding. The resultant conjugate, purified by gel filtration through Sephadex G-100, was characterised in terms of molecular size, charge properties and insulin content by polyacrylamide gel electrophoresis, agarose gel electrophoresis and immuno-dotblotting respectively. The conjugated protein was shown by gel band shift and nitrocellulose filter binding assays to bind DNA non-specifically in a reversible reaction which occurs rapidly, is dependent upon protein concentration and the ionic strength of the medium, and involves at least two types of intermolecular interaction. Furthermore, the conjugate was shown by competitive displacement of [ 125I ]insulin to bind specifically and particularly avidly to the HepG2 insulin receptor. When the expression vectors ptkNEO and pAL-8 which incorporate the neo gene were complexed to the conjugate in an in vitro transfection procedure using HepG2 cells, G418 resistant clones developed at frequencies of 2.0 - 5.5 X 10-5, possibly dependent upon vector promoter. Subsequently, a 923bp PstI fragment within the neD sequence was identified by Southern transfer in genomic DNA extracted from transfected cell populations which had been grown on a G418 regime through several subculture passages over a period of 44 days.