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.
Helmholtz Zentrum München, Technische Universität München, Institut für Entwicklungsgenetik, Ingolstädter Landstrasse 1, 85764 München, Neuherberg, Germany.
Laura
L
Schebelle
Claudia
C
Wolf
Carola
C
Stribl
Tahereh
T
Javaheri
Frank
F
Schnütgen
Andreas
A
Ettinger
Zoltán
Z
Ivics
Jens
J
Hansen
Patricia
P
Ruiz
Harald
H
von Melchner
Wolfgang
W
Wurst
Thomas
T
Floss
D000818:Animals; D002934:Cinnamates / Q000494:pharmacology; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis*; D018390:Gene Targeting / Q000379:methods*; D005819:Genetic Markers; D005822:Genetic Vectors; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D019426:Integrases / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic; D045522:Recombinases / Q000378:metabolism*; D011995:Recombination, Genetic; D019895:Transposases / Q000378:metabolism
D002934:Cinnamates; D018076:DNA, Complementary; D005819:Genetic Markers; D045522:Recombinases; D006921:Hygromycin B; C026273:hygromycin A; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases; D019895:Transposases; C494471:sleeping beauty transposase, human