In Vivo Hepatocyte Transduction with Retrovirus during In-flow Occlusion
| Autor: | D. G. Hafenrichter, Susan Kennedy, M. W. Flye, S. D. Rettinger, Saylors Rl, Katherine P. Ponder |
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| Rok vydání: | 1993 |
| Předmět: |
Male
Genetic enhancement medicine.medical_treatment Genetic Vectors Transfection Rats Sprague-Dawley Retrovirus In vivo Occlusion medicine Animals Hepatectomy biology Genetic Therapy beta-Galactosidase biology.organism_classification Molecular biology Liver Regeneration Rats Retroviridae medicine.anatomical_structure Liver Cell culture Hepatocyte Immunology Surgery Moloney murine leukemia virus Perfusion |
| Zdroj: | Journal of Surgical Research. 54:418-425 |
| ISSN: | 0022-4804 |
| DOI: | 10.1006/jsre.1993.1066 |
| Popis: | 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, retrovital supernatant was harvested from an amphotropic retroviral packaging cell line that packaged an LNL6-derived vector containing the cytomegaIovirus promoter driving expression of the Escherichia coli β-galactosidase (βgal) gene. Twenty-four hours after partial hepatectomy, experimental rats (N = 17) received 6 × 105 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-β- d -galactopyranoside to detect βgal 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 βgal. We conclude that retroviral transduction during in-flow occlusion is a rapid, reliable, low-mortality method of attaining stable genetic modification of regenerating rodent hepatocytes. |
| Databáze: | OpenAIRE |
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