Strong, Long-Term Transgene Expression in Rat Liver Using Chicken β-Actin Promoter Associated with Cytomegalovirus Immediate-Early Enhancer (CAG Promoter)
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 promote...
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doaj-5e47cc1832854bceb0e5b85ba5df4aaa2020-11-25T01:27:14ZengSAGE PublishingCell Transplantation0963-68971555-38922000-09-01910.1177/096368970000900513Strong, Long-Term Transgene Expression in Rat Liver Using Chicken β-Actin Promoter Associated with Cytomegalovirus Immediate-Early Enhancer (CAG Promoter)Motomichi Kosuga0Shin Enosawa1Xiao-Kang Li2Seiichi Suzuki3Nobutake Matsuo4Masao Yamada5Jayanta Roy-Chowdhury6Osamu Koiwai7Torayuki Okuyama8Department of Pediatrics, Keio University School of Medicine, Tokyo, JapanDepartments of Experimental Surgery, National Children's Medical Research Center, Tokyo, JapanDepartments of Experimental Surgery, National Children's Medical Research Center, Tokyo, JapanDepartments of Experimental Surgery, National Children's Medical Research Center, Tokyo, JapanDepartment of Pediatrics, Keio University School of Medicine, Tokyo, JapanDepartments of Genetics National Children's Medical Research Center, Tokyo, JapanDepartments of Medicine and Molecular Genetics, and the Marion Bessin Liver Research Center, Albert Einstein College of Medicine, New York, NYDepartment of Science and Technology, Science University of Tokyo, Tokyo, JapanDepartment of Pediatrics, Keio University School of Medicine, Tokyo, JapanFor 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-glucurono-syltransferase (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.https://doi.org/10.1177/096368970000900513 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Motomichi Kosuga Shin Enosawa Xiao-Kang Li Seiichi Suzuki Nobutake Matsuo Masao Yamada Jayanta Roy-Chowdhury Osamu Koiwai Torayuki Okuyama |
spellingShingle |
Motomichi Kosuga Shin Enosawa Xiao-Kang Li Seiichi Suzuki Nobutake Matsuo Masao Yamada Jayanta Roy-Chowdhury Osamu Koiwai Torayuki Okuyama Strong, Long-Term Transgene Expression in Rat Liver Using Chicken β-Actin Promoter Associated with Cytomegalovirus Immediate-Early Enhancer (CAG Promoter) Cell Transplantation |
author_facet |
Motomichi Kosuga Shin Enosawa Xiao-Kang Li Seiichi Suzuki Nobutake Matsuo Masao Yamada Jayanta Roy-Chowdhury Osamu Koiwai Torayuki Okuyama |
author_sort |
Motomichi Kosuga |
title |
Strong, Long-Term Transgene Expression in Rat Liver Using Chicken β-Actin Promoter Associated with Cytomegalovirus Immediate-Early Enhancer (CAG Promoter) |
title_short |
Strong, Long-Term Transgene Expression in Rat Liver Using Chicken β-Actin Promoter Associated with Cytomegalovirus Immediate-Early Enhancer (CAG Promoter) |
title_full |
Strong, Long-Term Transgene Expression in Rat Liver Using Chicken β-Actin Promoter Associated with Cytomegalovirus Immediate-Early Enhancer (CAG Promoter) |
title_fullStr |
Strong, Long-Term Transgene Expression in Rat Liver Using Chicken β-Actin Promoter Associated with Cytomegalovirus Immediate-Early Enhancer (CAG Promoter) |
title_full_unstemmed |
Strong, Long-Term Transgene Expression in Rat Liver Using Chicken β-Actin Promoter Associated with Cytomegalovirus Immediate-Early Enhancer (CAG Promoter) |
title_sort |
strong, long-term transgene expression in rat liver using chicken β-actin promoter associated with cytomegalovirus immediate-early enhancer (cag promoter) |
publisher |
SAGE Publishing |
series |
Cell Transplantation |
issn |
0963-6897 1555-3892 |
publishDate |
2000-09-01 |
description |
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-glucurono-syltransferase (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. |
url |
https://doi.org/10.1177/096368970000900513 |
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