Evidence for targeted gene transfer by receptor-mediated endocytosis. Stable expression following insulin-directed entry of NEO into HepG2 cells.
Evidence is presented for targeted gene delivery to HepG2 cells via the endocytotic pathway under the direction of insulin. Serum albumin was treated with the water-soluble carbodiimide N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and the resultant positively charged N-acylurea albumin covalently conjugated to insulin by glutaraldehyde cross-linkage. The conjugated protein is shown by nitrocellulose filter binding and gel band shift assays to bind DNA, and by competitive displacement of [125I]insulin to bind to the insulin receptor. When the expression vectors ptkNEO and pAL-8 which incorporate the neo gene were complexed to the conjugate in an in vitro system of transfection, G418 resistant clones developed at frequencies of 2.0-5.5 x 10(-5). Subsequently, a 923bp PstI fragment within the neo sequence was identified by Southern transfer in genomic DNA from transfected cell populations which had been maintained on a G418 regime for 44 days.
Huckett
B
B
Department of Biochemistry, University of Durban-Westville, South Africa.
Ariatti
M
M
Hawtrey
A O
AO
D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004251:DNA Transposable Elements; D004705:Endocytosis; D007328:Insulin / Q000378:metabolism*; D011972:Receptor, Insulin / Q000502:physiology*; D012709:Serum Albumin / Q000378:metabolism; D014162:Transfection
D004251:DNA Transposable Elements; D007328:Insulin; D012709:Serum Albumin; D004247:DNA; D011972:Receptor, Insulin