Fate of DNA targeted to the liver by asialoglycoprotein receptor-mediated endocytosis in vivo: Prolonged persistence in cytoplasmic vesicles after partial hepatectomy

Namita Roy Chowdhury, Catherine H. Wu, George Y. Wu, Purna C. Yerneni, Vasudeva R. Bommineni, Jayanta Roy-Chowdhury

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Abstract

After intravenous injection, DNA complexed with asialoglycoprotein-polylysine conjugates is endocytosed by hepatocytes via asialoglycoprotein receptors and is expressed transiently. Long term persistence and expression occurs when partial hepatectomy is performed after gene delivery. To determine the intracellular location of the persisting DNA, we transferred a plasmid expressing bacterial chloramphenicol acetyltransferase into the liver of rats in vivo by asialoglycoprotein receptor-mediated endocytosis. The internalized DNA was measured by Southern blot. Twenty min after administration, 80-85% of the plasmid appeared in the liver, 80% of which was within hepatocytes (12,000-18,000 copies/hepatocyte). In sham-operated control rats, the transgene concentration decreased to 8-12 and 2-4% of the initial levels in 4 and 24 h, respectively, and became undetectable at 7 days. In rats subjected to 66% hepatectomy 20 min after DNA administration, 20, 9, and 7% of the plasmid in the residual liver persisted at 4 h, 24 h, and 7 days, respectively. Liver homogenates were fractionated by differential centrifugation and Percoll gradient centrifugation. In 66% hepatectomized rats, the plasmid persisted in an undegraded, transfection-competent form in plasma membrane/endosome-enriched fractions throughout the duration of the experiment (7 days), indicating that cytoplasmic vesicles are the main site of persistence of the endocytosed DNA.

Original languageEnglish (US)
Pages (from-to)11265-11271
Number of pages7
JournalJournal of Biological Chemistry
Volume268
Issue number15
StatePublished - May 25 1993

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Asialoglycoprotein Receptor
Cytoplasmic Vesicles
Hepatectomy
Endocytosis
Liver
Plasmids
DNA
Rats
Hepatocytes
Centrifugation
Rat control
Chloramphenicol O-Acetyltransferase
Endosomes
Cell membranes
Southern Blotting
Transgenes
Intravenous Injections
Transfection
Genes
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry

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Fate of DNA targeted to the liver by asialoglycoprotein receptor-mediated endocytosis in vivo : Prolonged persistence in cytoplasmic vesicles after partial hepatectomy. / Chowdhury, Namita Roy; Wu, Catherine H.; Wu, George Y.; Yerneni, Purna C.; Bommineni, Vasudeva R.; Roy-Chowdhury, Jayanta.

In: Journal of Biological Chemistry, Vol. 268, No. 15, 25.05.1993, p. 11265-11271.

Research output: Contribution to journalArticle

Chowdhury, Namita Roy ; Wu, Catherine H. ; Wu, George Y. ; Yerneni, Purna C. ; Bommineni, Vasudeva R. ; Roy-Chowdhury, Jayanta. / Fate of DNA targeted to the liver by asialoglycoprotein receptor-mediated endocytosis in vivo : Prolonged persistence in cytoplasmic vesicles after partial hepatectomy. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 15. pp. 11265-11271.
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abstract = "After intravenous injection, DNA complexed with asialoglycoprotein-polylysine conjugates is endocytosed by hepatocytes via asialoglycoprotein receptors and is expressed transiently. Long term persistence and expression occurs when partial hepatectomy is performed after gene delivery. To determine the intracellular location of the persisting DNA, we transferred a plasmid expressing bacterial chloramphenicol acetyltransferase into the liver of rats in vivo by asialoglycoprotein receptor-mediated endocytosis. The internalized DNA was measured by Southern blot. Twenty min after administration, 80-85{\%} of the plasmid appeared in the liver, 80{\%} of which was within hepatocytes (12,000-18,000 copies/hepatocyte). In sham-operated control rats, the transgene concentration decreased to 8-12 and 2-4{\%} of the initial levels in 4 and 24 h, respectively, and became undetectable at 7 days. In rats subjected to 66{\%} hepatectomy 20 min after DNA administration, 20, 9, and 7{\%} of the plasmid in the residual liver persisted at 4 h, 24 h, and 7 days, respectively. Liver homogenates were fractionated by differential centrifugation and Percoll gradient centrifugation. In 66{\%} hepatectomized rats, the plasmid persisted in an undegraded, transfection-competent form in plasma membrane/endosome-enriched fractions throughout the duration of the experiment (7 days), indicating that cytoplasmic vesicles are the main site of persistence of the endocytosed DNA.",
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