title: In vivo hepatocyte transduction with retrovirus during in-flow occlusion.
abstract: Gene therapy research would be facilitated by a technically simple procedure for transducing hepatocytes in vivo. Previously reported methods have employed partial hepatectomy followed 24 hr later by asanguineous perfusion of the regenerating liver with retrovirus. We have developed a simpler method of in vivo transduction in which we deliver an intraportal bolus of retrovirus to the regenerating rodent liver during a brief period of hepatic in-flow occlusion. On Day 0, adult male Sprague-Dawley rats (N = 19) underwent 70% hepatectomy to induce hepatocyte replication. On Day 1, retroviral supernatant was harvested from an amphotropic retroviral packaging cell line that packaged an LNL6-derived vector containing the cytomegalovirus promoter driving expression of the Escherichia coli beta-galactosidase (beta gal) gene. Twenty-four hours after partial hepatectomy, experimental rats (N = 17) received 6 x 10(5) colony-forming units of retrovirus by intraportal injection during a 3-min occlusion of the hepatic artery and portal vein. Control rats (N = 2) received intraportal medium (without retrovirus), also during in-flow occlusion. The procedure required 20-25 min, and the survival rate was 84%. Cryostat sections were prepared from liver biopsies obtained on Post-transduction Days 8 and 15 and stained with 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside to detect beta gal expression. Light microscopic examination of Day 8 sections from surviving experimental rats (N = 14) revealed 0.10-1.00% blue (i.e., transduced) hepatocytes per low power field, while sections from control rats (N = 2) exhibited no blue cells. Day 15 sections from experimental rats revealed a somewhat lower frequency of hepatocytes expressing beta gal.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: S D
authors.initials: SD
authors.lastname: Rettinger
authors.forename: K P
authors.initials: KP
authors.lastname: Ponder
authors.forename: R L
authors.initials: RL
authors.lastname: Saylors
authors.forename: S C
authors.initials: SC
authors.lastname: Kennedy
authors.forename: D G
authors.initials: DG
authors.lastname: Hafenrichter
authors.forename: M W
authors.initials: MW
authors.lastname: Flye
mesh_terms: D000818:Animals; D015316:Genetic Therapy; D005822:Genetic Vectors; D006498:Hepatectomy; D008099:Liver / Q000378:metabolism* / Q000502:physiology; D008115:Liver Regeneration; D008297:Male; D008979:Moloney murine leukemia virus / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D001616:beta-Galactosidase