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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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Schematic overview of the efficiency of FLPo-mediated RMCE in FlEx gene trap ES cell clones. Shown are all transfected gene trap clone insertions and the corresponding rounded efficiency of exchange reaction of pEx-FLP-dsRed in three different reading frames (0 upper, +1 middle and +2 lowest values in percent, red values indicating the frame matching the gene trap insertion). For the Tardbp insertion, the exchange vector pEx-FLP-hTDP-43(A315T) was used. All gene trap clones used had insertions in an intron of the respective gene, and the ratio on the right indicates in which intron out of how many total introns the insertion is located. On the left, the size of the genes is indicated in kilobases, and the total size of the genes is not drawn in scale, with omitted DNA indicated. Untranslated and translated exons according to Ensembl. MGI Gene Identifiers and absolute expression levels in ES cells are listed in Supplementary Table S1.
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Figure 2: Schematic overview of the efficiency of FLPo-mediated RMCE in FlEx gene trap ES cell clones. Shown are all transfected gene trap clone insertions and the corresponding rounded efficiency of exchange reaction of pEx-FLP-dsRed in three different reading frames (0 upper, +1 middle and +2 lowest values in percent, red values indicating the frame matching the gene trap insertion). For the Tardbp insertion, the exchange vector pEx-FLP-hTDP-43(A315T) was used. All gene trap clones used had insertions in an intron of the respective gene, and the ratio on the right indicates in which intron out of how many total introns the insertion is located. On the left, the size of the genes is indicated in kilobases, and the total size of the genes is not drawn in scale, with omitted DNA indicated. Untranslated and translated exons according to Ensembl. MGI Gene Identifiers and absolute expression levels in ES cells are listed in Supplementary Table S1.

Mentions: With the exception of Tardbp and Gtf2ird insertions (Figure 2), gene trap clones with insertions of rFLPRosabetageo or rsFRosabetageo were chosen randomly in either 0, +1 or +2 reading frames. rsFRosabetageo consists of a splice acceptor α-geo polyA cassette, flanked on each side by FRT, F3, loxP and lox5171 or lox511, in head-to-head orientation (FlEx array, Figure 1B). For further details, see http://www.genetrap.de.Figure 2.


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)

Schematic overview of the efficiency of FLPo-mediated RMCE in FlEx gene trap ES cell clones. Shown are all transfected gene trap clone insertions and the corresponding rounded efficiency of exchange reaction of pEx-FLP-dsRed in three different reading frames (0 upper, +1 middle and +2 lowest values in percent, red values indicating the frame matching the gene trap insertion). For the Tardbp insertion, the exchange vector pEx-FLP-hTDP-43(A315T) was used. All gene trap clones used had insertions in an intron of the respective gene, and the ratio on the right indicates in which intron out of how many total introns the insertion is located. On the left, the size of the genes is indicated in kilobases, and the total size of the genes is not drawn in scale, with omitted DNA indicated. Untranslated and translated exons according to Ensembl. MGI Gene Identifiers and absolute expression levels in ES cells are listed in Supplementary Table S1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Schematic overview of the efficiency of FLPo-mediated RMCE in FlEx gene trap ES cell clones. Shown are all transfected gene trap clone insertions and the corresponding rounded efficiency of exchange reaction of pEx-FLP-dsRed in three different reading frames (0 upper, +1 middle and +2 lowest values in percent, red values indicating the frame matching the gene trap insertion). For the Tardbp insertion, the exchange vector pEx-FLP-hTDP-43(A315T) was used. All gene trap clones used had insertions in an intron of the respective gene, and the ratio on the right indicates in which intron out of how many total introns the insertion is located. On the left, the size of the genes is indicated in kilobases, and the total size of the genes is not drawn in scale, with omitted DNA indicated. Untranslated and translated exons according to Ensembl. MGI Gene Identifiers and absolute expression levels in ES cells are listed in Supplementary Table S1.
Mentions: With the exception of Tardbp and Gtf2ird insertions (Figure 2), gene trap clones with insertions of rFLPRosabetageo or rsFRosabetageo were chosen randomly in either 0, +1 or +2 reading frames. rsFRosabetageo consists of a splice acceptor α-geo polyA cassette, flanked on each side by FRT, F3, loxP and lox5171 or lox511, in head-to-head orientation (FlEx array, Figure 1B). For further details, see http://www.genetrap.de.Figure 2.

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