Efficient conditional and promoter-specific in vivo expression of cDNAs of choice by taking advantage of recombinase-mediated cassette exchange using FlEx gene traps

  • Recombinase-mediated cassette exchange (RMCE) exploits the possibility to unidirectionally exchange any genetic material flanked by heterotypic recombinase recognition sites (RRS) with target sites in the genome. Due to a limited number of available pre-fabricated target sites, RMCE in mouse embryonic stem (ES) cells has not been tapped to its full potential to date. Here, we introduce a universal system, which allows the targeted insertion of any given transcriptional unit into 85 742 previously annotated retroviral conditional gene trap insertions, representing 7013 independent genes in mouse ES cells, by RMCE. This system can be used to express any given cDNA under the control of endogenous trapped promoters in vivo, as well as for the generation of transposon ‘launch pads’ for chromosomal region-specific ‘Sleeping Beauty’ insertional mutagenesis. Moreover, transcription of the gene-of-interest is only activated upon Cre-recombinase activity, a feature that adds conditionality to this expression system, which is demonstrated in vivo. The use of the RMCE system presented in this work requires one single-cloning step followed by one overnight gateway clonase reaction and subsequent cassette exchange in ES cells with efficiencies of 40% in average.

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Author:Laura Schebelle, Claudia Wolf, Carola Stribl, Tahereh Javaheri, Frank SchnütgenORCiDGND, Andreas Ettinger, Zoltán Ivics, Jens Hansen, Patricia Ruiz, Harald von Melchner, Wolfgang WurstORCiD, Thomas Floss
Pubmed Id:https://pubmed.ncbi.nlm.nih.gov/20139417
Parent Title (English):Nucleic acids research
Publisher:Oxford Univ. Press
Place of publication:Oxford
Document Type:Article
Date of Publication (online):2010/11/12
Date of first Publication:2010/05/01
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2010/11/12
Issue:9, e106
Page Number:12
First Page:1
Last Page:12
© The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Source:Nucleic Acids Research, (2010) 38 (9): e106 ; doi: 10.1093/nar/gkq044
Institutes:Medizin / Medizin
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Sammlung Biologie / Sondersammelgebiets-Volltexte
Licence (German):License LogoCreative Commons - Namensnennung-Nicht kommerziell 3.0