Strong, long-term transgene expression in rat liver using chicken β-Actin promoter associated with cytomegalovirus immediate-early enhancer (CAG promoter)

Motomichi Kosuga, Shin Enosawa, Xiao Kang Li, Seiichi Suzuki, Nobutake Matsuo, Masao Yamada, Jayanta Roy-Chowdhury, Osamu Koiwai, Torayuki Okuyama

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20 Scopus citations

Abstract

For successful gene therapy in hepatic enzyme deficiencies, it is essential to use promoters that can maintain strong transcriptional activity for the long term in the liver. Using Gunn rats, a model animal for Crigler-Najjar syndrome type I, the long-term transcriptional function of the CAG promoter (a combination of chicken β-actin promoter and cytomegalovirus immediate-early enhancer) was evaluated in the rat liver. We constructed a plasmid pCAGGHUGT, containing expression cassettes of human bilirubin UDP-glucuronosyltransferase (BUGT) and hygromycin phosphotransferase, under the control of the CAG promoter and murine phosphoglycerate kinase promoter, respectively. Conditionally immortalized Gunn rat hepatocytes (IGRH), which had been established using mutant SV40 large T antigen ((TS)T), were transfected with pCAGGHUGT. A stably transfected clone IGRHUGT, expressing a high level of BUGT, was obtained after selection with hygromycin. At 33°C, the cells doubled in number in approximately 72 h; however, at 37°C, cell proliferation stopped, indicating that the characteristic of temperature-dependent proliferation was retained in this clone. Ten million cells were injected into the spleen of syngeneic Gunn rats five times at 10-day intervals. Serum bilirubin levels were reduced by 45-50% at 70 days after the first transplantation and remained so throughout the duration of the study (120 days). These results suggested that the CAG promoter was able to maintain strong transcriptional activity in rat liver for at least 120 days.

Original languageEnglish (US)
Pages (from-to)675-680
Number of pages6
JournalCell Transplantation
Volume9
Issue number5
DOIs
Publication statusPublished - Jan 1 2000

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Keywords

  • CAG promoter
  • Gunn rats
  • Immortalized hepatocytes

ASJC Scopus subject areas

  • Biomedical Engineering
  • Cell Biology
  • Transplantation

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