Efficient Conditional and Promoter-specific in Vivo Expression of CDNAs of Choice by Taking Advantage of Recombinase-mediated Cassette Exchange Using FlEx Gene Traps

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2010 Feb 9

PMID 20139417

Citations 15

Authors

Laura Schebelle

Claudia Wolf

Carola Stribl

Tahereh Javaheri

Frank Schnutgen

Andreas Ettinger

Zoltan Ivics

Jens Hansen

Patricia Ruiz

Harald von Melchner

Wolfgang Wurst

Thomas Floss

Affiliations

Soon will be listed here.

Abstract

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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