title: Inducible and reversible lentiviral and Recombination Mediated Cassette Exchange (RMCE) systems for controlling gene expression.
abstract: Manipulation of gene expression to invoke loss of function (LoF) or gain of function (GoF) phenotypes is important for interrogating complex biological questions both in vitro and in vivo. Doxycycline (Dox)-inducible gene expression systems are commonly used although success is often limited by high background and insufficient sensitivity to Dox. Here we develop broadly applicable platforms for reliable, tightly controlled and reversible Dox-inducible systems for lentiviral mediated generation of cell lines or FLP Recombination-Mediated Cassette Exchange (RMCE) into the Collagen 1a1 (Col1a1) locus (FLP-In Col1a1) in mouse embryonic stem cells. We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs. We demonstrate the usefulness of these platforms in ectopic gene expression or gene knockdown in multiple cell lines, primary neurons and in FLP-In Col1a1 mouse embryonic stem cells. We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs. These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function.
authors.affiliation: School of Molecular and Biomedical Science (Biochemistry), The University of Adelaide, Adelaide, South Australia, Australia; Institute of Molecular Pathology, The University of Adelaide, Adelaide, South Australia, Australia.
authors.forename: David C
authors.initials: DC
authors.lastname: Bersten
authors.affiliation: School of Molecular and Biomedical Science (Biochemistry), The University of Adelaide, Adelaide, South Australia, Australia; Institute of Molecular Pathology, The University of Adelaide, Adelaide, South Australia, Australia.
authors.forename: Adrienne E
authors.initials: AE
authors.lastname: Sullivan
authors.affiliation: School of Molecular and Biomedical Science (Biochemistry), The University of Adelaide, Adelaide, South Australia, Australia; Institute of Molecular Pathology, The University of Adelaide, Adelaide, South Australia, Australia.
authors.forename: Dian
authors.initials: D
authors.lastname: Li
authors.affiliation: School of Molecular and Biomedical Science (Biochemistry), The University of Adelaide, Adelaide, South Australia, Australia; Institute of Molecular Pathology, The University of Adelaide, Adelaide, South Australia, Australia.
authors.forename: Veronica
authors.initials: V
authors.lastname: Bhakti
authors.affiliation: School of Molecular and Biomedical Science (Genetics), The University of Adelaide, Adelaide, South Australia, Australia; Robinson Research Institute, The University of Adelaide, Adelaide, South Australia, Australia.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Bent
authors.affiliation: School of Molecular and Biomedical Science (Biochemistry), The University of Adelaide, Adelaide, South Australia, Australia; Institute of Molecular Pathology, The University of Adelaide, Adelaide, South Australia, Australia.
authors.forename: Murray L
authors.initials: ML
authors.lastname: Whitelaw
mesh_terms: D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D024042:Collagen Type I / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D018076:DNA, Complementary / Q000097:blood / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D018014:Gene Transfer Techniques; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D016254:Mutagenesis, Insertional / Q000379:methods; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic
chemical_list: D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D018076:DNA, Complementary; D034741:RNA, Small Interfering; D004318:Doxycycline