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.
Highlights
Control of gene expression in mammalian cells and in animal models is of significant importance to basic and applied biological research
Modular, and all-inclusive Dox-inducible expression systems delivered through lentiviral infection or Recombination Mediated Cassette Exchange (RMCE), which overcome many previous limitations
We demonstrate improvements in background expression, sensitivity, and shRNA screening, which allow for efficient generation of reliable gain or loss of gene expression systems
Summary
Control of gene expression in mammalian cells and in animal models is of significant importance to basic and applied biological research. Doxycycline (Dox)-inducible expression systems have been used widely for ectopic expression and knockdown of genes both in cultured cells and in vivo, significant challenges concerning the reliability of expression in cell lines or animals and significant background expression have limited their use. Site-specific insertion of inducible microRNA-30 context (miR30c) based shRNA cassettes in embryonic stem cells have enabled rapid generation of mice with inducible gene knockdown [1,2]. These systems employ Tet activators driven from different genomic sites, do not provide expression in all tissues, and lack a flexible platform to gain tissue-specific expression. The algorithms used to predict potent miR30c flanked shRNA sequences able to knockdown gene expression from singly integrated constructs are yet to be fully developed, time-consuming testing of multiple sequences for each target gene of interest is still required
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