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

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.

Prenatal diagnosis of bilirubin-UDP-glucuronosyltransferase deficiency in rats by genomic DNA analysis.

Hepatic bilirubin excretion requires UDP-glucuronosyltransferase-mediated glucuronidation. Patients with type I Crigler-Najjar syndrome and mutant rats (Gunn strain) inherit deficiency of UDP-glucuronyltransferase activity toward bilirubin as an autosomal recessive trait and, as a result, exhibit marked nonhemolytic unconjugated hyperbilirubinemia throughout postnatal life. Heterozygous carriers of the trait have normal serum bilirubin levels. Because of placental excretion of unconjugated bilirubin, type 1 Crigler-Najjar syndrome patients and Gunn rats are not jaundiced in utero, making prenatal diagnosis difficult. Here we report a diagnostic method in Gunn rats based on genomic DNA analysis for prenatal recognition of deficiency of UDP-glucuronyltransferase activity toward bilirubin in Gunn rats and identification of heterozygous carriers. We and others have shown that two distinct messenger RNA species (UDP-glucuronyltransferase activity toward bilirubin and the 3-methylcholanthrene-inducible phenol-UDP-glucuronyltransferase messenger RNA) in Gunn rat liver contain identical deletions of a single guanosine residue in their common 3' regions. Loss of the restriction site for the endonuclease BstNI, which results from this deletion, was used as the basis for a diagnostic test. Female heterozygous Gunn rats were mated with male homozygous Gunn rats. Genomic DNA was extracted from the chorionic aspect of placenta of 17-day fetuses or from leukocytes from normal rats, obligate heterozygotes and homozygous Gunn rats. The DNA was sequentially digested with the restriction enzymes EcoRI and BstNI and subjected to Southern-blot analysis with a double-stranded DNA probe for the common region of UDP-glucuronyltransferase activity toward bilirubin and the 3-methylcholanthrene-inducible UDP-glucuronyltransferase messenger RNAs.(ABSTRACT TRUNCATED AT 250 WORDS)