Limits...
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

Crossing scheme for generating EGFP tagged MiMIC lines.Females carrying the hs-FLP and vasa-phiC31 integrase on the X-chromosome, and the FRT flanked multiple tag (GFSTF) cassette on the II-chromosome are crossed to males carrying a MiMIC insertion in a coding intron of a gene on the III-chromosome. The resulting embryos are heat shocked, and adult progeny with the desired genotype are crossed with flies carrying appropriate balancers. The resulting progeny is screened for the loss of white+ and yellow+ and crossed to flies carrying appropriate balancers to establish stocks, which are then verified by PCR assay.DOI:http://dx.doi.org/10.7554/eLife.08469.005
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4499919&req=5

fig2: Crossing scheme for generating EGFP tagged MiMIC lines.Females carrying the hs-FLP and vasa-phiC31 integrase on the X-chromosome, and the FRT flanked multiple tag (GFSTF) cassette on the II-chromosome are crossed to males carrying a MiMIC insertion in a coding intron of a gene on the III-chromosome. The resulting embryos are heat shocked, and adult progeny with the desired genotype are crossed with flies carrying appropriate balancers. The resulting progeny is screened for the loss of white+ and yellow+ and crossed to flies carrying appropriate balancers to establish stocks, which are then verified by PCR assay.DOI:http://dx.doi.org/10.7554/eLife.08469.005

Mentions: To initiate RMCE, we crossed the appropriate donor flies to MiMIC-containing flies and heat shocked the resulting embryos and larvae (Figure 2). Within the primordial germ cells of some of the embryos and larvae, phiC31 integrase catalyzed recombination between attB sites in the donor and attP sites in the MiMIC transposon. The positive RMCE events were selected based on the loss of the y + marker present in the original MiMIC (Figure 2). We confirmed the integration and orientation of the donor cassette by PCR as described in Venken et al. (2011). Typically, 50% of the integration events are in the proper orientation.10.7554/eLife.08469.005Figure 2.Crossing scheme for generating EGFP tagged MiMIC lines.


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)

Crossing scheme for generating EGFP tagged MiMIC lines.Females carrying the hs-FLP and vasa-phiC31 integrase on the X-chromosome, and the FRT flanked multiple tag (GFSTF) cassette on the II-chromosome are crossed to males carrying a MiMIC insertion in a coding intron of a gene on the III-chromosome. The resulting embryos are heat shocked, and adult progeny with the desired genotype are crossed with flies carrying appropriate balancers. The resulting progeny is screened for the loss of white+ and yellow+ and crossed to flies carrying appropriate balancers to establish stocks, which are then verified by PCR assay.DOI:http://dx.doi.org/10.7554/eLife.08469.005
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499919&req=5

fig2: Crossing scheme for generating EGFP tagged MiMIC lines.Females carrying the hs-FLP and vasa-phiC31 integrase on the X-chromosome, and the FRT flanked multiple tag (GFSTF) cassette on the II-chromosome are crossed to males carrying a MiMIC insertion in a coding intron of a gene on the III-chromosome. The resulting embryos are heat shocked, and adult progeny with the desired genotype are crossed with flies carrying appropriate balancers. The resulting progeny is screened for the loss of white+ and yellow+ and crossed to flies carrying appropriate balancers to establish stocks, which are then verified by PCR assay.DOI:http://dx.doi.org/10.7554/eLife.08469.005
Mentions: To initiate RMCE, we crossed the appropriate donor flies to MiMIC-containing flies and heat shocked the resulting embryos and larvae (Figure 2). Within the primordial germ cells of some of the embryos and larvae, phiC31 integrase catalyzed recombination between attB sites in the donor and attP sites in the MiMIC transposon. The positive RMCE events were selected based on the loss of the y + marker present in the original MiMIC (Figure 2). We confirmed the integration and orientation of the donor cassette by PCR as described in Venken et al. (2011). Typically, 50% of the integration events are in the proper orientation.10.7554/eLife.08469.005Figure 2.Crossing scheme for generating EGFP tagged MiMIC lines.

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