Doxycycline -inducible expression 4-PPBP maleate systems have been used widely for ectopic expression and knockdown of genes both in cultured cells and in vivo, however significant challenges concerning the reliability of expression in cell lines or animals and significant background expression have limited their use. Recently, site-specific insertion of inducible microRNA-30 context based shRNA cassettes in embryonic stem cells have enabled rapid generation of mice with inducible gene knockdown. However, these systems employ Tet activators driven fromdifferent genomic sites, do not provide expression in all tissues, and lack a flexible platform to gain tissue-specific expression. Impaired Dox-inducible expression in some tissues may be due to lower promoter activity in these tissues, decreased accessibility of the transcriptional machinery to the loci expressing either the Tet-activator or the Tetresponsive factor in certain tissues, or limited delivery of Dox to these tissues. In addition, the algorithms used to predict potentmiR30c flanked shRNA sequences able to knockdown gene expression from singly integrated constructs are yet to be fully developed, thus time-consuming testing of multiple sequences for each target gene of interest is still required. Here we generate flexible and reliable Dox-inducible expression systems which can be rapidly employed for GoF and LoF, overcoming difficulties previously encountered with Dox-inducible expression systems. We designed and generated Gateway-compatible lentiviral or Recombination Mediated Cassette Exchange vectors, which contain all the components necessary to gain strong Dox-inducible expression froma near-zero background.We developed amodular platform, which can easily be adjusted to gain tissue-specific expression, and use alternative Tetracycline activators to increase the sensitivity to Dox, which should overcome poor response in some tissues due to limited delivery of Dox.We also designed and tested a miR30c shRNA prediction tool, which enables effective knockdown of target genes from singly integrated Dox-inducible cassettes. These platforms provide a broadly applicable 3PO Tet-On system and enable improved methods for rapid generation of cell lines and mice in order to study gain and loss of gene function. Here we have engineered optimised, broadly applicable platforms for generation of Doxinducible expression in immortalised cell lines, primary neurons and use with RMCE at a specific site in embryonic stem cells. Using a 3rd generation Tet-activator system we have created lentiviral constructs capable of generating near homogenous Dox-inducible cell lines free of background expression and capable of strong and reversible ectopic expression or gene knockdown. We have also engineered a flexible Flp-In RMCE system for generation of site-specific