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A genetic toolkit for tagging intronic MiMIC containing genes.

Nagarkar-Jaiswal S, DeLuca SZ, Lee PT, Lin WW, Pan H, Zuo Z, Lv J, Spradling AC, Bellen HJ - Elife (2015)

Bottom Line: These modified genes permit numerous applications including assessment of protein expression pattern, identification of protein interaction partners by immunoprecipitation followed by mass spec, and reversible removal of the tagged protein in any tissue.At present, these conversions remain time and labor-intensive as they require embryos to be injected with plasmid DNA containing the exon tag.We document the efficiency and tag 60 mostly uncharacterized genes.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States.

ABSTRACT
Previously, we described a large collection of Minos-Mediated Integration Cassettes (MiMICs) that contain two phiC31 recombinase target sites and allow the generation of a new exon that encodes a protein tag when the MiMIC is inserted in a codon intron (Nagarkar-Jaiswal et al., 2015). These modified genes permit numerous applications including assessment of protein expression pattern, identification of protein interaction partners by immunoprecipitation followed by mass spec, and reversible removal of the tagged protein in any tissue. At present, these conversions remain time and labor-intensive as they require embryos to be injected with plasmid DNA containing the exon tag. In this study, we describe a simple and reliable genetic strategy to tag genes/proteins that contain MiMIC insertions using an integrated exon encoding GFP flanked by FRT sequences. We document the efficiency and tag 60 mostly uncharacterized genes.

No MeSH data available.


Related in: MedlinePlus

Schematic of flippase and phiC31 integrase mediated in vivo protein tagging.(A) Flp mediated generation of circular DNA with tag sequences. Donor line carries a source of Flp recombinase and phiC31 integrase, and the core cassette for a specific phase (0, 1, or 2) encoding a (GGS)4 flexible linker (L), multiple tags EGFP-FlAsH-StrepII-TEVcs-3xFlag (GFSTF), another (GGS)4 flexible linker, and a splice donor (SD), which is flanked by two inverted attB and two FRT sites oriented in that same direction. Upon Flp expression, the core is flipped out as a circular DNA containing one FRT site. (B) In vivo integration of tag sequence into Minos-Mediated Integration Cassette (MiMIC) locus. At a MiMIC locus in a coding intron, the MiMIC gene trap cassette is replaced by L-EGFP-FlAsH-StrepII-TEVcs-3xFlag-L sequence by phiC31 integrase resulting in loss of yellow+ marker.DOI:http://dx.doi.org/10.7554/eLife.08469.00210.7554/eLife.08469.003Figure 1—source data 1.List of constructs.DOI:http://dx.doi.org/10.7554/eLife.08469.003List of fly strains generated.Stocks #1 to 6 are stocks that contain the GFSTF construct (phase 0, 1, and 2) for tagging genes/proteins that map to chromosome II and III. Stocks # 7: FRT cassette maps to chromosome 2 and contains phase 0 construct; #8: FRT cassette maps to chromosome 2 and contains phase 1 construct; #9: FRT cassette maps to chromosome 2 and contains phase 2 construct; #10: FRT cassette maps to chromosome 3 and contains phase 0 construct; #11: FRT cassette maps to chromosome 3 and contains phase 1 construct; #12 FRT cassette maps to chromosome 3 and contains phase 2 construct.DOI:http://dx.doi.org/10.7554/eLife.08469.004
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fig1: Schematic of flippase and phiC31 integrase mediated in vivo protein tagging.(A) Flp mediated generation of circular DNA with tag sequences. Donor line carries a source of Flp recombinase and phiC31 integrase, and the core cassette for a specific phase (0, 1, or 2) encoding a (GGS)4 flexible linker (L), multiple tags EGFP-FlAsH-StrepII-TEVcs-3xFlag (GFSTF), another (GGS)4 flexible linker, and a splice donor (SD), which is flanked by two inverted attB and two FRT sites oriented in that same direction. Upon Flp expression, the core is flipped out as a circular DNA containing one FRT site. (B) In vivo integration of tag sequence into Minos-Mediated Integration Cassette (MiMIC) locus. At a MiMIC locus in a coding intron, the MiMIC gene trap cassette is replaced by L-EGFP-FlAsH-StrepII-TEVcs-3xFlag-L sequence by phiC31 integrase resulting in loss of yellow+ marker.DOI:http://dx.doi.org/10.7554/eLife.08469.00210.7554/eLife.08469.003Figure 1—source data 1.List of constructs.DOI:http://dx.doi.org/10.7554/eLife.08469.003List of fly strains generated.Stocks #1 to 6 are stocks that contain the GFSTF construct (phase 0, 1, and 2) for tagging genes/proteins that map to chromosome II and III. Stocks # 7: FRT cassette maps to chromosome 2 and contains phase 0 construct; #8: FRT cassette maps to chromosome 2 and contains phase 1 construct; #9: FRT cassette maps to chromosome 2 and contains phase 2 construct; #10: FRT cassette maps to chromosome 3 and contains phase 0 construct; #11: FRT cassette maps to chromosome 3 and contains phase 1 construct; #12 FRT cassette maps to chromosome 3 and contains phase 2 construct.DOI:http://dx.doi.org/10.7554/eLife.08469.004

Mentions: We developed a genetic strategy that allows the desired RMCE event to take place efficiently without the need for microinjection. The method uses FLP recombinase to release a genomically integrated DNA flanked by FRT sites into the nucleoplasm where it can efficiently undergo phiC31 integrase-mediated cassette exchange, as shown by Gohl et al. (2011). As shown in Figure 1A, we engineered three donor cassettes, one for each reading frame. The core, which contains a splice acceptor (SA) followed by a (GGS)4 flexible linker, multiple tags (EGFP-FlAsH-StrepII-TEVcs-3xFlag {GFSTF}), another (GGS)4 flexible linker, and a splice donor (SD), is flanked by two inverted attB sites for phiC31-mediated RMCE (Venken et al., 2011). We then cloned this cassette core between tandem FRT sites in a P-element transformation vector (Gong and Golic, 2003). FLP-mediated recombination between the tandem FRT sites excises a circular donor DNA molecule from its initial genetic locus, promoting its efficient recombination with a distal target site (Golic et al., 1997). A mini-white eye color marker gene between our donor cassette and one of the FRT sites allows us to monitor the presence or absence of the donor cassette in FLP recombinase-containing stocks.10.7554/eLife.08469.002Figure 1.Schematic of flippase and phiC31 integrase mediated in vivo protein tagging.


A genetic toolkit for tagging intronic MiMIC containing genes.

Nagarkar-Jaiswal S, DeLuca SZ, Lee PT, Lin WW, Pan H, Zuo Z, Lv J, Spradling AC, Bellen HJ - Elife (2015)

Schematic of flippase and phiC31 integrase mediated in vivo protein tagging.(A) Flp mediated generation of circular DNA with tag sequences. Donor line carries a source of Flp recombinase and phiC31 integrase, and the core cassette for a specific phase (0, 1, or 2) encoding a (GGS)4 flexible linker (L), multiple tags EGFP-FlAsH-StrepII-TEVcs-3xFlag (GFSTF), another (GGS)4 flexible linker, and a splice donor (SD), which is flanked by two inverted attB and two FRT sites oriented in that same direction. Upon Flp expression, the core is flipped out as a circular DNA containing one FRT site. (B) In vivo integration of tag sequence into Minos-Mediated Integration Cassette (MiMIC) locus. At a MiMIC locus in a coding intron, the MiMIC gene trap cassette is replaced by L-EGFP-FlAsH-StrepII-TEVcs-3xFlag-L sequence by phiC31 integrase resulting in loss of yellow+ marker.DOI:http://dx.doi.org/10.7554/eLife.08469.00210.7554/eLife.08469.003Figure 1—source data 1.List of constructs.DOI:http://dx.doi.org/10.7554/eLife.08469.003List of fly strains generated.Stocks #1 to 6 are stocks that contain the GFSTF construct (phase 0, 1, and 2) for tagging genes/proteins that map to chromosome II and III. Stocks # 7: FRT cassette maps to chromosome 2 and contains phase 0 construct; #8: FRT cassette maps to chromosome 2 and contains phase 1 construct; #9: FRT cassette maps to chromosome 2 and contains phase 2 construct; #10: FRT cassette maps to chromosome 3 and contains phase 0 construct; #11: FRT cassette maps to chromosome 3 and contains phase 1 construct; #12 FRT cassette maps to chromosome 3 and contains phase 2 construct.DOI:http://dx.doi.org/10.7554/eLife.08469.004
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fig1: Schematic of flippase and phiC31 integrase mediated in vivo protein tagging.(A) Flp mediated generation of circular DNA with tag sequences. Donor line carries a source of Flp recombinase and phiC31 integrase, and the core cassette for a specific phase (0, 1, or 2) encoding a (GGS)4 flexible linker (L), multiple tags EGFP-FlAsH-StrepII-TEVcs-3xFlag (GFSTF), another (GGS)4 flexible linker, and a splice donor (SD), which is flanked by two inverted attB and two FRT sites oriented in that same direction. Upon Flp expression, the core is flipped out as a circular DNA containing one FRT site. (B) In vivo integration of tag sequence into Minos-Mediated Integration Cassette (MiMIC) locus. At a MiMIC locus in a coding intron, the MiMIC gene trap cassette is replaced by L-EGFP-FlAsH-StrepII-TEVcs-3xFlag-L sequence by phiC31 integrase resulting in loss of yellow+ marker.DOI:http://dx.doi.org/10.7554/eLife.08469.00210.7554/eLife.08469.003Figure 1—source data 1.List of constructs.DOI:http://dx.doi.org/10.7554/eLife.08469.003List of fly strains generated.Stocks #1 to 6 are stocks that contain the GFSTF construct (phase 0, 1, and 2) for tagging genes/proteins that map to chromosome II and III. Stocks # 7: FRT cassette maps to chromosome 2 and contains phase 0 construct; #8: FRT cassette maps to chromosome 2 and contains phase 1 construct; #9: FRT cassette maps to chromosome 2 and contains phase 2 construct; #10: FRT cassette maps to chromosome 3 and contains phase 0 construct; #11: FRT cassette maps to chromosome 3 and contains phase 1 construct; #12 FRT cassette maps to chromosome 3 and contains phase 2 construct.DOI:http://dx.doi.org/10.7554/eLife.08469.004
Mentions: We developed a genetic strategy that allows the desired RMCE event to take place efficiently without the need for microinjection. The method uses FLP recombinase to release a genomically integrated DNA flanked by FRT sites into the nucleoplasm where it can efficiently undergo phiC31 integrase-mediated cassette exchange, as shown by Gohl et al. (2011). As shown in Figure 1A, we engineered three donor cassettes, one for each reading frame. The core, which contains a splice acceptor (SA) followed by a (GGS)4 flexible linker, multiple tags (EGFP-FlAsH-StrepII-TEVcs-3xFlag {GFSTF}), another (GGS)4 flexible linker, and a splice donor (SD), is flanked by two inverted attB sites for phiC31-mediated RMCE (Venken et al., 2011). We then cloned this cassette core between tandem FRT sites in a P-element transformation vector (Gong and Golic, 2003). FLP-mediated recombination between the tandem FRT sites excises a circular donor DNA molecule from its initial genetic locus, promoting its efficient recombination with a distal target site (Golic et al., 1997). A mini-white eye color marker gene between our donor cassette and one of the FRT sites allows us to monitor the presence or absence of the donor cassette in FLP recombinase-containing stocks.10.7554/eLife.08469.002Figure 1.Schematic of flippase and phiC31 integrase mediated in vivo protein tagging.

Bottom Line: These modified genes permit numerous applications including assessment of protein expression pattern, identification of protein interaction partners by immunoprecipitation followed by mass spec, and reversible removal of the tagged protein in any tissue.At present, these conversions remain time and labor-intensive as they require embryos to be injected with plasmid DNA containing the exon tag.We document the efficiency and tag 60 mostly uncharacterized genes.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States.

ABSTRACT
Previously, we described a large collection of Minos-Mediated Integration Cassettes (MiMICs) that contain two phiC31 recombinase target sites and allow the generation of a new exon that encodes a protein tag when the MiMIC is inserted in a codon intron (Nagarkar-Jaiswal et al., 2015). These modified genes permit numerous applications including assessment of protein expression pattern, identification of protein interaction partners by immunoprecipitation followed by mass spec, and reversible removal of the tagged protein in any tissue. At present, these conversions remain time and labor-intensive as they require embryos to be injected with plasmid DNA containing the exon tag. In this study, we describe a simple and reliable genetic strategy to tag genes/proteins that contain MiMIC insertions using an integrated exon encoding GFP flanked by FRT sequences. We document the efficiency and tag 60 mostly uncharacterized genes.

No MeSH data available.


Related in: MedlinePlus