A novel conditional ZsGreen-expressing transgenic reporter rat strain for validating Cre recombinase expression

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Abstract

The Cre/ loxP recombination system has revolutionized the ability to genetically manipulate animal genomes in order to conditionally control gene expression. With recent advances in genome editing, barriers to manipulating the rat genome have been overcome and it is now possible to generate new rat strains (Cre drivers) in which Cre recombinase expression is carefully controlled temporally and/or spatially. However, the ability to evaluate and characterize these Cre driver strains is limited by the availability of reliable reporter rat strains. Here, we describe the generation and characterization of a new transgenic rat strain in which conditional expression of the ZsGreen fluorescent protein gene requires the presence of exogenous Cre recombinase. Breeding Cre-expressing rat strains to this stable ZsGreen reporter strain provides an ideal method for validating new rat Cre driver lines and will greatly accelerate the characterization pipeline.

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Recombinase-driver rat lines: tools, techniques, and optogenetic application to dopamine-mediated reinforcement.

Ilana B. Witten, Elizabeth E. Steinberg, Soo-Yeun Lee … (2011)

Currently there is no general approach for achieving specific optogenetic control of genetically defined cell types in rats, which provide a powerful experimental system for numerous established neurophysiological and behavioral paradigms. To overcome this challenge we have generated genetically restricted recombinase-driver rat lines suitable for driving gene expression in specific cell types, expressing Cre recombinase under the control of large genomic regulatory regions (200-300 kb). Multiple tyrosine hydroxylase (Th)::Cre and choline acetyltransferase (Chat)::Cre lines were produced that exhibited specific opsin expression in targeted cell types. We additionally developed methods for utilizing optogenetic tools in freely moving rats and leveraged these technologies to clarify the causal relationship between dopamine (DA) neuron firing and positive reinforcement, observing that optical stimulation of DA neurons in the ventral tegmental area (VTA) of Th::Cre rats is sufficient to support vigorous intracranial self-stimulation (ICSS). These studies complement existing targeting approaches by extending the generalizability of optogenetics to traditionally non-genetically-tractable but vital animal models. Copyright © 2011 Elsevier Inc. All rights reserved.

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Binary recombinase systems for high-resolution conditional mutagenesis

Mario Hermann, Patrick Stillhard, Hendrik Wildner … (2014)

Conditional mutagenesis using Cre recombinase expressed from tissue specific promoters facilitates analyses of gene function and cell lineage tracing. Here, we describe two novel dual-promoter-driven conditional mutagenesis systems designed for greater accuracy and optimal efficiency of recombination. Co-Driver employs a recombinase cascade of Dre and Dre-respondent Cre, which processes loxP-flanked alleles only when both recombinases are expressed in a predetermined temporal sequence. This unique property makes Co-Driver ideal for sequential lineage tracing studies aimed at unraveling the relationships between cellular precursors and mature cell types. Co-InCre was designed for highly efficient intersectional conditional transgenesis. It relies on highly active trans-splicing inteins and promoters with simultaneous transcriptional activity to reconstitute Cre recombinase from two inactive precursor fragments. By generating native Cre, Co-InCre attains recombination rates that exceed all other binary SSR systems evaluated in this study. Both Co-Driver and Co-InCre significantly extend the utility of existing Cre-responsive alleles.

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Conditional gene expression in the mouse inner ear using Cre-loxP.

Brandon C Cox, Zhiyong Liu, Marcia Lagarde … (2012)

In recent years, there has been significant progress in the use of Cre-loxP technology for conditional gene expression in the inner ear. Here, we introduce the basic concepts of this powerful technology, emphasizing the differences between Cre and CreER. We describe the creation and Cre expression pattern of each Cre and CreER mouse line that has been reported to have expression in auditory and vestibular organs. We compare the Cre expression patterns between Atoh1-CreER(TM) and Atoh1-CreER(T2) and report a new line, Fgfr3-iCreER(T2), which displays inducible Cre activity in cochlear supporting cells. We also explain how results can vary when transgenic vs. knock-in Cre/CreER alleles are used to alter gene expression. We discuss practical issues that arise when using the Cre-loxP system, such as the use of proper controls, Cre efficiency, reporter expression efficiency, and Cre leakiness. Finally, we introduce other methods for conditional gene expression, including Flp recombinase and the tetracycline-inducible system, which can be combined with Cre-loxP mouse models to investigate conditional expression of more than one gene.

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Author and article information

Contributors

Elizabeth C. Bryda: brydae@missouri.edu

Journal

Journal ID (nlm-ta): Sci Rep

Journal ID (iso-abbrev): Sci Rep

Title: Scientific Reports

Publisher: Nature Publishing Group UK (London )

ISSN (Electronic): 2045-2322

Publication date (Electronic): 16 September 2019

Publication date PMC-release: 16 September 2019

Publication date Collection: 2019

Volume: 9

Electronic Location Identifier: 13330

Affiliations

[1 ]ISNI 0000 0001 2162 3504, GRID grid.134936.a, Rat Resource and Research Center, , University of Missouri, ; Columbia, Missouri United States of America

[2 ]ISNI 0000 0001 2162 3504, GRID grid.134936.a, Department of Veterinary Pathobiology, College of Veterinary Medicine, , University of Missouri, ; Columbia, Missouri United States of America

[3 ]ISNI 0000 0001 2162 3504, GRID grid.134936.a, Animal Modeling Core, , University of Missouri, ; Columbia, Missouri United States of America

[4 ]ISNI 0000 0001 2162 3504, GRID grid.134936.a, Comparative Medicine Program, College of Veterinary Medicine, , University of Missouri, ; Columbia, Missouri United States of America

Article

Publisher ID: 49783

DOI: 10.1038/s41598-019-49783-w

PMC ID: 6746712

PubMed ID: 31527612

SO-VID: 019f1b9a-8880-46cd-b39f-2933a4e28fa6

License:

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 18 January 2019

Date accepted : 27 August 2019

Funding

Funded by: FundRef https://doi.org/10.13039/100000002, U.S. Department of Health & Human Services | National Institutes of Health (NIH);

Award ID: P40 OD011062

Award Recipient : Hongsheng Men

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ScienceOpen disciplines: Uncategorized

Keywords: genetic engineering,gene expression

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ScienceOpen disciplines: Uncategorized

Keywords: genetic engineering, gene expression

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A novel conditional ZsGreen-expressing transgenic reporter rat strain for validating Cre recombinase expression

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Most cited references 11

Recombinase-driver rat lines: tools, techniques, and optogenetic application to dopamine-mediated reinforcement.

Binary recombinase systems for high-resolution conditional mutagenesis

Conditional gene expression in the mouse inner ear using Cre-loxP.

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