Flexible Accelerated STOP Tetracycline Operator-Knockin (FAST): A Versatile and Efficient New Gene Modulating System

Abstract

We created the FAST (Flexible Accelerated STOP TetO-knockin) system, an efficient method for manipulating gene expression in vivo to rapidly screen animal models of disease. A single gene targeting event yields 2 distinct knockin mice -STOP-tetO and tetO knockin-which permit generation of multiple strains with variable expression patterns: 1) knockout, 2) Cre-mediated rescue; 3) tTA-mediated misexpression; 4) tTA-mediated overexpression; and 5) tTS-mediated conditional knockout/knockdown. Using the FAST system, multiple gain-and loss-of-function strains can therefore be generated on a timescale not previously achievable. These strains can then be screened for clinically-relevant abnormalities. We demonstrate the flexibility and broad applicability of the FAST system by targeting several genes encoding proteins implicated in neuropsychiatric disorders: Mlc1, Neuroligin 3, the serotonin 1A receptor, and the serotonin 1B receptor. Keywordsgenetics; gene targeting; animal model; mouse; conditional modulation of gene expression; developmental change Gain-of-function and loss-of-function studies are commonly used to examine gene function in vivo, particularly in attempts to model human disease in animals. Developing animal models of disease is key to the process of elucidating neuropsychiatric disease pathophysiology, in turn leading to drug discovery and translation to patient populations. Financial DisclosureAll authors declare that they have no biomedical financial interests and no potential conflicts of interest.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Fig 1A). It allows us to take advantage of established Cre-recombinase, tTA (tetracycline-controlled transcriptional-activator), and tTS (tetracycline-controlled transcriptional-silencer) lines to rapidly produce 5 separate lines of mice from the original knock-in: 1) knockout; 2) Cremediated rescue; 3) tTA-mediated ectopic expression; 4) tTA-mediated overexpression; and 5) tTS-mediated conditional knockout/knockdown. NIH Public AccessThe FAST system allows us therefore to rapidly generate multiple lines of mice that provide a spectrum of expression levels for single genes, from selective knockout to selective overexpression. In addition, the FAST system has the added advantage of easily integrating temporal and spatial specificity into the manipulations of gene expression. In this paper, we demonstrate the efficacy of the FAST system using multiple genes implicated in neuropsychiatric disorders. One of our overall goals is to use the FAST system to make mouse models using genes that have been linked to dise...

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Genetic and early environmental influences on the serotonin system: consequences for brain development and risk for psychopathology

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