Optimized self-excising Cre-expression cassette for mammalian cells

Biochemical and Biophysical Research Communications
Anssi J MähönenSeppo Ylä-Herttuala

Abstract

We observed that overexpression of Cre recombinase in 293 T cells has toxic effects and that the chicken beta-actin promoter is active in Escherichia coli, causing expression of Cre in bacteria. This led to significant problems in the cloning of Cre/loxP constructs. Leaky Cre-expression in E. coli, and toxicity of the Cre overexpression in mammalian cells, were solved by constructing a novel silent self-inactivating Cre (SSi-Cre) expression cassette. The SSi-Cre is based on modified loxP sites flanking the Cre/Int/DsRed fusion gene containing a Cre coding sequence interrupted by an intron, which prevents leaky expression of Cre in E. coli. Additionally, this system contains a reporter gene to visualize Cre activity by fluorescent microscopy. The SSi-Cre cassette provides a universal strategy for the generation of Cre/loxP constructs, as well as a solution to the toxicity caused by the overexpression of Cre in target cells. SSi-Cre should thus provide a useful tool for various applications based on the Cre/loxP system.

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Citations

Jun 21, 2007·Nature Immunology·Marc Schmidt-Supprian, Klaus Rajewsky
May 26, 2012·Circulation Research·Thomas Doetschman, Mohamad Azhar
Apr 19, 2006·American Journal of Respiratory Cell and Molecular Biology·Jeffrey A Whitsett, Anne-Karina T Perl
May 19, 2011·Yi chuan = Hereditas·Fu-Yu Lin, Xiao Yang
Dec 7, 2013·The Journal of Pathology·Esther RodriguezScott K Lyons
Feb 11, 2014·Neuron·Karine LoulierJean Livet
Nov 28, 2013·Cell Cycle·Vaibhao C JanbandhuReinhard Fässler
Feb 28, 2019·American Journal of Physiology. Renal Physiology·Rohan S BalkawadeCaroline B Marshall
Apr 22, 2009·The Journal of Immunology : Official Journal of the American Association of Immunologists·Atsuko Yoshioka HigashiMotoko Yanagita
Dec 4, 2021·Molecular Therapy : the Journal of the American Society of Gene Therapy·Jingwei SongMichael Michaelides

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