title: A predictable ligand regulated expression strategy for stably integrated transgenes in mammalian cells in culture.
abstract: 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.
authors.affiliation: BIOTEC, TU Dresden, c/o Max-Planck-Institut für Molekulare Zellbiologie und Genetik MPI-CBG, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Anastassiadis
authors.forename: Jinhyun
authors.initials: J
authors.lastname: Kim
authors.forename: Nathalie
authors.initials: N
authors.lastname: Daigle
authors.forename: Rolf
authors.initials: R
authors.lastname: Sprengel
authors.forename: Hans R
authors.initials: HR
authors.lastname: Schöler
authors.forename: A Francis
authors.initials: AF
authors.lastname: Stewart
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D005786:Gene Expression Regulation / Q000235:genetics*; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011944:Receptors, Androgen / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D019076:Transgenes / Q000235:genetics*
chemical_list: D017405:Herpes Simplex Virus Protein Vmw65; D008024:Ligands; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein