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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) Removal of hygromycin resistance upon Cre activity. Cre-transfected exchange clone A03 (E307D01/pExFLP-dsRed derivative) was analysed using primers located within both the hygromycin/dsRed cassettes (TP) and the LTR of the original gene trap vector (SR). PCR on total genomic DNA of five pooled transfected dishes (lanes 1–5) compared to genomic DNA of clone A03 before Cre transfection (A03 −Cre). The internal primer (TP) gives rise to the larger product (619 bp) after successful excision of the hygromycin cassette. Before hygromycin excision, primer TP amplifies a smaller product of 239 bp. (B) RT-PCR and triple PCR analysis to determine splicing to the dsRed after hygromycin excision by using external primers in exon II (5) of the insertion locus (Msi2) and an internal dsRed primer (I) and as internal wild-type control a primer located in exon III (3) of the Msi2 gene. cDNA of individual clones derived from Cre-transfected clone A03 after puromycin selection (lanes labelled 1–3) compared to cDNA of clone A03 before Cre transfection (lane A03 −Cre). As control genomic DNA of two gene trap clones (E307D01 and E326E12) and pEX-FLP-dsRed plasmid was used. (C) Western blot analysis of protein extracted from ES cells probed with a polyclonal RFP antibody. As controls wild-type ES cells either untransfected (WT) or transiently transfected with two different dsRed expression plasmids (WT + RFPI + II) were used. Exchange clones A03 and D04 were analysed prior (−CRE) and after (+CRE) transient CRE transfection. DsRed protein positive samples show a 27-kDa band. Lower blot shows loading control with beta actin monoclonal antibody (42 kDa).
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Figure 3: (A) Removal of hygromycin resistance upon Cre activity. Cre-transfected exchange clone A03 (E307D01/pExFLP-dsRed derivative) was analysed using primers located within both the hygromycin/dsRed cassettes (TP) and the LTR of the original gene trap vector (SR). PCR on total genomic DNA of five pooled transfected dishes (lanes 1–5) compared to genomic DNA of clone A03 before Cre transfection (A03 −Cre). The internal primer (TP) gives rise to the larger product (619 bp) after successful excision of the hygromycin cassette. Before hygromycin excision, primer TP amplifies a smaller product of 239 bp. (B) RT-PCR and triple PCR analysis to determine splicing to the dsRed after hygromycin excision by using external primers in exon II (5) of the insertion locus (Msi2) and an internal dsRed primer (I) and as internal wild-type control a primer located in exon III (3) of the Msi2 gene. cDNA of individual clones derived from Cre-transfected clone A03 after puromycin selection (lanes labelled 1–3) compared to cDNA of clone A03 before Cre transfection (lane A03 −Cre). As control genomic DNA of two gene trap clones (E307D01 and E326E12) and pEX-FLP-dsRed plasmid was used. (C) Western blot analysis of protein extracted from ES cells probed with a polyclonal RFP antibody. As controls wild-type ES cells either untransfected (WT) or transiently transfected with two different dsRed expression plasmids (WT + RFPI + II) were used. Exchange clones A03 and D04 were analysed prior (−CRE) and after (+CRE) transient CRE transfection. DsRed protein positive samples show a 27-kDa band. Lower blot shows loading control with beta actin monoclonal antibody (42 kDa).

Mentions: To minimize secondary effects, the selection marker of pEX-FLP vector was designed to be removable by Cre recombinase in vitro or in vivo. To demonstrate excision in vitro, clone A03 was transiently transfected with Caggs-Cre-IRES-Puro plasmid with and without subsequent puromycin selection. Genomic DNA of plates without selection were pooled and screened by PCR with primers located within the splice acceptor sequence (hygromycin and dsRed) and in the LTR. This PCR yielded different-sized products before (239 bp) or after (619 bp) excision of the selection marker (Figure 3A). Lanes marked 1–5 (pooled transfected plates) show both band sizes, whereas untransfected A03 only shows the smaller product. Without puromycin selection, Cre excision occurred only partially. To ensure the splicing to the dsRed cassette after Cre excision, the same transfection was performed followed by puromycin selection and cDNA of individual clones was screened by PCR with primers located in Msi2 exon II and an internal primer in the dsRed coding sequence (Figure 3B). This primer set yielded a 550-bp product in three independent clones (lanes 1–3), which was sequence-verified. As an internal control, a third primer located in Msi2 exon III was added, which yielded a 94-bp product of the wild-type transcript. As further controls, genomic DNA of two gene trap clones (E307D01 and E326E12) and the plasmid DNA of pEX-FLP were used. Gene trap genomic DNA templates yielded the wild-type genomic product (427 bp) including the intron 3/4.


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) Removal of hygromycin resistance upon Cre activity. Cre-transfected exchange clone A03 (E307D01/pExFLP-dsRed derivative) was analysed using primers located within both the hygromycin/dsRed cassettes (TP) and the LTR of the original gene trap vector (SR). PCR on total genomic DNA of five pooled transfected dishes (lanes 1–5) compared to genomic DNA of clone A03 before Cre transfection (A03 −Cre). The internal primer (TP) gives rise to the larger product (619 bp) after successful excision of the hygromycin cassette. Before hygromycin excision, primer TP amplifies a smaller product of 239 bp. (B) RT-PCR and triple PCR analysis to determine splicing to the dsRed after hygromycin excision by using external primers in exon II (5) of the insertion locus (Msi2) and an internal dsRed primer (I) and as internal wild-type control a primer located in exon III (3) of the Msi2 gene. cDNA of individual clones derived from Cre-transfected clone A03 after puromycin selection (lanes labelled 1–3) compared to cDNA of clone A03 before Cre transfection (lane A03 −Cre). As control genomic DNA of two gene trap clones (E307D01 and E326E12) and pEX-FLP-dsRed plasmid was used. (C) Western blot analysis of protein extracted from ES cells probed with a polyclonal RFP antibody. As controls wild-type ES cells either untransfected (WT) or transiently transfected with two different dsRed expression plasmids (WT + RFPI + II) were used. Exchange clones A03 and D04 were analysed prior (−CRE) and after (+CRE) transient CRE transfection. DsRed protein positive samples show a 27-kDa band. Lower blot shows loading control with beta actin monoclonal antibody (42 kDa).
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Related In: Results  -  Collection

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Figure 3: (A) Removal of hygromycin resistance upon Cre activity. Cre-transfected exchange clone A03 (E307D01/pExFLP-dsRed derivative) was analysed using primers located within both the hygromycin/dsRed cassettes (TP) and the LTR of the original gene trap vector (SR). PCR on total genomic DNA of five pooled transfected dishes (lanes 1–5) compared to genomic DNA of clone A03 before Cre transfection (A03 −Cre). The internal primer (TP) gives rise to the larger product (619 bp) after successful excision of the hygromycin cassette. Before hygromycin excision, primer TP amplifies a smaller product of 239 bp. (B) RT-PCR and triple PCR analysis to determine splicing to the dsRed after hygromycin excision by using external primers in exon II (5) of the insertion locus (Msi2) and an internal dsRed primer (I) and as internal wild-type control a primer located in exon III (3) of the Msi2 gene. cDNA of individual clones derived from Cre-transfected clone A03 after puromycin selection (lanes labelled 1–3) compared to cDNA of clone A03 before Cre transfection (lane A03 −Cre). As control genomic DNA of two gene trap clones (E307D01 and E326E12) and pEX-FLP-dsRed plasmid was used. (C) Western blot analysis of protein extracted from ES cells probed with a polyclonal RFP antibody. As controls wild-type ES cells either untransfected (WT) or transiently transfected with two different dsRed expression plasmids (WT + RFPI + II) were used. Exchange clones A03 and D04 were analysed prior (−CRE) and after (+CRE) transient CRE transfection. DsRed protein positive samples show a 27-kDa band. Lower blot shows loading control with beta actin monoclonal antibody (42 kDa).
Mentions: To minimize secondary effects, the selection marker of pEX-FLP vector was designed to be removable by Cre recombinase in vitro or in vivo. To demonstrate excision in vitro, clone A03 was transiently transfected with Caggs-Cre-IRES-Puro plasmid with and without subsequent puromycin selection. Genomic DNA of plates without selection were pooled and screened by PCR with primers located within the splice acceptor sequence (hygromycin and dsRed) and in the LTR. This PCR yielded different-sized products before (239 bp) or after (619 bp) excision of the selection marker (Figure 3A). Lanes marked 1–5 (pooled transfected plates) show both band sizes, whereas untransfected A03 only shows the smaller product. Without puromycin selection, Cre excision occurred only partially. To ensure the splicing to the dsRed cassette after Cre excision, the same transfection was performed followed by puromycin selection and cDNA of individual clones was screened by PCR with primers located in Msi2 exon II and an internal primer in the dsRed coding sequence (Figure 3B). This primer set yielded a 550-bp product in three independent clones (lanes 1–3), which was sequence-verified. As an internal control, a third primer located in Msi2 exon III was added, which yielded a 94-bp product of the wild-type transcript. As further controls, genomic DNA of two gene trap clones (E307D01 and E326E12) and the plasmid DNA of pEX-FLP were used. Gene trap genomic DNA templates yielded the wild-type genomic product (427 bp) including the intron 3/4.

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