Elsevier

Biochemical Pharmacology

Volume 40, Issue 2, 15 July 1990, Pages 253-263
Biochemical Pharmacology

Evidence for targeted gene transfer by receptor-mediated endocytosis: Stable expression following insulin-directed entry of neo into HepG2 cells

https://doi.org/10.1016/0006-2952(90)90686-FGet rights and content

Abstract

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 × 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.

References (46)

  • E.M. Southern

    Detection of specific sequences among DNA fragments separated by gel electrophoresis

    J Mol Biol

    (1975)
  • S. Marshall

    Kinetics of insulin receptor internalisation and recycling in adipocytes: shunting of receptors to a degradative pathway by inhibitors of recycling

    J Biol Chem

    (1985)
  • F. Colbere-Garapin et al.

    A new dominant hybrid selective marker for higher eukaryotic cells

    J Mol Biol

    (1981)
  • S.G. Rogers et al.

    Gene transfer in plants: production of transformed plants using Ti plasmid vectors

    Methods Enzymol

    (1986)
  • G.J. Darlington

    Liver cell lines

    Methods Enzymol

    (1987)
  • G.Y. Wu et al.

    Receptor-mediated in vitro gene transformation by a soluble DNA carrier system

    J Biol Chem

    (1987)
  • G.Y. Wu et al.

    Receptor-mediated gene delivery and expression in vivo

    J Biol Chem

    (1988)
  • M.J. Gething et al.

    Cell surface expression of influenza haemagglutinin from a cloned DNA copy of the RNA gene

    Nature

    (1981)
  • B. Hjelle et al.

    Oncogene v-src transforms and establishes embryonic rodent fibroblast but not diploid human fibroblast

  • R. Jaenisch

    Transgenic animals

    Science

    (1988)
  • F. Constantini et al.

    Correction of murine β-thalassemia by gene transfer into the germ line

    Science

    (1986)
  • F.L. Graham et al.

    Transformation of mammalian cells with DNA using the calcium technique

  • S. Kawai et al.

    New procedure for DNA transfection with polycation and dimethyl sulfoxide

    Mol Cell Biol

    (1984)
  • Cited by (0)

    Present address: Department of Pharmacology, Faculty of Medicine, University of Stellenbosch, P.O. Box 63, Tygerberg 7505, South Africa.

    View full text