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| ID:
15622.0,
MPI für molekulare Zellbiologie und Genetik / Publikationen mpi-cbg 2003 |
| A predictable ligand regulated expression strategy for stably integrated transgenes in mammalian cells in culture |
| Authors: | Anastassiadis, K.; Kim, J.; Daigle, N.; Prengel, R.; Schöler, H. R.; Stewart, A. F. | | Language: | English | | Date of Publication (YYYY-MM-DD): | 2002-10-02 | | Title of Journal: | Gene | | Journal Abbrev.: | Gene | | Volume: | 298 | | Issue / Number: | 2 | | Start Page: | 159 | | End Page: | 172 | | Review Status: | Peer-review | | Audience: | Not Specified | | Abstract / Description: | Several strategies for regulated stable transgene expression in mammalian cells have been described. These strategies have different strengths and weaknesses, however they all share a common problem, namely predictability in application. Here we address this problem using the leading strategy for ligand inducible transgene expression, the tetracycline repressor system. Initially, we found the best stable clone out of 48 examined showed only 6-fold inducibility. Hence we looked for additions and modifications that improve the chances of a successful outcome. We document three important aspects; first, use of a mammalian codon-optimized tetracycline repressor gene; second, addition of a steroid hormone receptor ligand binding domain to the tetracycline repressor-virion protein 16 fusion protein activator; third, flanking the tet-operator/transgene cassette with insulator elements from the chicken beta-globin locus. By inclusion of these three design features, 18/18 clones showed low basal and highly inducible (>50 X) expression. (C) 2002 Elsevier Science B.V. All rights reserved. | | Free Keywords: | codon improved reverse tetracycline repressor fused to virion protein 16 activation domain (rtTA); rtTA-ligand binding domain transactivator; fusions; insulators | | Comment of the Author/Creator: | Date: 2002, OCT 2 | | External Publication Status: | published | | Document Type: | Article |
| Communicated by: | N. N. | | Affiliations: | MPI für molekulare Zellbiologie und Genetik
| | External Affiliations: | Tech Univ Dresden, BIOTEC, D-01062 Dresden, Germany; Max Planck Inst Med Res, Dept Mol Neurobiol, D-69120 Heidelberg, Germany; EMBL, Gene Express Program, D-69117 Heidelberg, Germany; Univ Penn, Sch Vet Med, Ctr Anim Transgenesis & Germ Cell Res, New Bolton Ctr, Kennett Sq, PA 19348, USA
| | Identifiers: | ISI:000179435800006 [ID No:1]
ISSN:0378-1119 [ID No:2] | |
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