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

Schebelle L, Wolf C, Stribl C, Javaheri T, Schnütgen F, Ettinger A, Ivics Z, Hansen J, Ruiz P, von Melchner H, Wurst W, Floss T - Nucleic Acids Res. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Helmholtz Zentrum München, Technische Universität München, Institut für Entwicklungsgenetik, Ingolstädter Landstrasse 1, 85764 München, Neuherberg, Germany.

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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(A) Schematic outline of the procedure for individual modification and use of pEX-FLP. Any DNA sequence, which is cloned between the attL1/2 recombination sites in the pENTR-EX, can be exchanged with an attR1/2 flanked cat ccdb cassette of pEX-Dest in vitro. (B) Schematic illustration of the FLPo RMCE in ES cells. pEX-FLP harbors two heterospecific flippase recombination sites, therefore cotransfection of FLPo and pEX-FLP to any FlEx gene trap ES cell clone allows replacement of β-geo cassette. (C) Analysis of successfully exchanged locus with pEX-FLP-dsRed containing a adSA-dsRed-BGHpA sequence flanked by IR/DR of Sleeping Beauty transposase. A representative PCR screen of seven hygromycin resistant clones (E307D01 derivates, A01–A07) concerning orientation of the genetrap vector and successful exchange (left: primers B045, B048 and B050; right: primers SR, TP). A01 and A06 are inverted β-geo insertions and show an 800-bp band on the left and no band on the right. A02–A05 are successfully exchanged clones and show an 839-bp band on the left and the small 239-bp band on the right. Clone A07 carries the original β-geo insertion and gives rise to the 631-bp band on the left and the 516-bp band on the right.
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Figure 1: (A) Schematic outline of the procedure for individual modification and use of pEX-FLP. Any DNA sequence, which is cloned between the attL1/2 recombination sites in the pENTR-EX, can be exchanged with an attR1/2 flanked cat ccdb cassette of pEX-Dest in vitro. (B) Schematic illustration of the FLPo RMCE in ES cells. pEX-FLP harbors two heterospecific flippase recombination sites, therefore cotransfection of FLPo and pEX-FLP to any FlEx gene trap ES cell clone allows replacement of β-geo cassette. (C) Analysis of successfully exchanged locus with pEX-FLP-dsRed containing a adSA-dsRed-BGHpA sequence flanked by IR/DR of Sleeping Beauty transposase. A representative PCR screen of seven hygromycin resistant clones (E307D01 derivates, A01–A07) concerning orientation of the genetrap vector and successful exchange (left: primers B045, B048 and B050; right: primers SR, TP). A01 and A06 are inverted β-geo insertions and show an 800-bp band on the left and no band on the right. A02–A05 are successfully exchanged clones and show an 839-bp band on the left and the small 239-bp band on the right. Clone A07 carries the original β-geo insertion and gives rise to the 631-bp band on the left and the 516-bp band on the right.

Mentions: Between the 3′ loxP and the F3 site, a unique PmeI restriction site was used to insert a blunt-end Gateway cassette A (Invitrogen) containing an attR 1/2 flanked ccdB cassette (16) to finalize pEx-Dest (Figure 1).Figure 1.


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

Schebelle L, Wolf C, Stribl C, Javaheri T, Schnütgen F, Ettinger A, Ivics Z, Hansen J, Ruiz P, von Melchner H, Wurst W, Floss T - Nucleic Acids Res. (2010)

(A) Schematic outline of the procedure for individual modification and use of pEX-FLP. Any DNA sequence, which is cloned between the attL1/2 recombination sites in the pENTR-EX, can be exchanged with an attR1/2 flanked cat ccdb cassette of pEX-Dest in vitro. (B) Schematic illustration of the FLPo RMCE in ES cells. pEX-FLP harbors two heterospecific flippase recombination sites, therefore cotransfection of FLPo and pEX-FLP to any FlEx gene trap ES cell clone allows replacement of β-geo cassette. (C) Analysis of successfully exchanged locus with pEX-FLP-dsRed containing a adSA-dsRed-BGHpA sequence flanked by IR/DR of Sleeping Beauty transposase. A representative PCR screen of seven hygromycin resistant clones (E307D01 derivates, A01–A07) concerning orientation of the genetrap vector and successful exchange (left: primers B045, B048 and B050; right: primers SR, TP). A01 and A06 are inverted β-geo insertions and show an 800-bp band on the left and no band on the right. A02–A05 are successfully exchanged clones and show an 839-bp band on the left and the small 239-bp band on the right. Clone A07 carries the original β-geo insertion and gives rise to the 631-bp band on the left and the 516-bp band on the right.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875000&req=5

Figure 1: (A) Schematic outline of the procedure for individual modification and use of pEX-FLP. Any DNA sequence, which is cloned between the attL1/2 recombination sites in the pENTR-EX, can be exchanged with an attR1/2 flanked cat ccdb cassette of pEX-Dest in vitro. (B) Schematic illustration of the FLPo RMCE in ES cells. pEX-FLP harbors two heterospecific flippase recombination sites, therefore cotransfection of FLPo and pEX-FLP to any FlEx gene trap ES cell clone allows replacement of β-geo cassette. (C) Analysis of successfully exchanged locus with pEX-FLP-dsRed containing a adSA-dsRed-BGHpA sequence flanked by IR/DR of Sleeping Beauty transposase. A representative PCR screen of seven hygromycin resistant clones (E307D01 derivates, A01–A07) concerning orientation of the genetrap vector and successful exchange (left: primers B045, B048 and B050; right: primers SR, TP). A01 and A06 are inverted β-geo insertions and show an 800-bp band on the left and no band on the right. A02–A05 are successfully exchanged clones and show an 839-bp band on the left and the small 239-bp band on the right. Clone A07 carries the original β-geo insertion and gives rise to the 631-bp band on the left and the 516-bp band on the right.
Mentions: Between the 3′ loxP and the F3 site, a unique PmeI restriction site was used to insert a blunt-end Gateway cassette A (Invitrogen) containing an attR 1/2 flanked ccdB cassette (16) to finalize pEx-Dest (Figure 1).Figure 1.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Helmholtz Zentrum München, Technische Universität München, Institut für Entwicklungsgenetik, Ingolstädter Landstrasse 1, 85764 München, Neuherberg, Germany.

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.

Show MeSH
Related in: MedlinePlus