A predictable ligand regulated expression strategy for stably integrated transgenes in mammalian cells in culture

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 li...

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Bibliographic Details
Published in:Gene 2002-10, Vol.298 (2), p.159-172
Main Authors: Anastassiadis, Konstantinos, Kim, Jinhyun, Daigle, Nathalie, Sprengel, Rolf, Schöler, Hans R, Stewart, A Francis
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Base Sequence
Binding Sites - genetics
Cell Line
Gene Expression Regulation - genetics
Herpes Simplex Virus Protein Vmw65 - genetics
Humans
Ligands
Molecular Sequence Data
Plasmids - genetics
Receptors, Androgen - genetics
Recombinant Fusion Proteins - genetics
Repressor Proteins - genetics
Transgenes - genetics
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Summary: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.
ISSN:0378-1119
1879-0038
DOI:10.1016/s0378-1119(02)00979-4